Chapter INTRODUCTION. Spawning induction of red tilapia (Oreochromis niloticus) through reproductive

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1 Chapter INTRODUCTION Spawning induction of red tilapia (Oreochromis niloticus) through reproductive manipulation has been carried out by many researchers in an effort to increase tilapia production for the aquaculture industry. There is an increasing commercial demand for tilapia fish from year to year worldwide due to health awareness through eating fish. For example, in 2003, it has been reported that the Malaysia fish market for tilapia was 22,560 metric tonnes (Lim, 2006). Due to increase in demand, natural reproduction is not able to cope with the high production. Therefore, to achieve higher tilapia production, hormonal induction technique may be one of the techniques that may need to be applied to speed up the spawning events in order to achieve higher production. Understanding the role of the neuroendocrine system in the regulation of the reproductive system is essential in order to manipulate spawning. Application of endocrinebased technology by administration of exogenous spawning hormone is part of the strategy to achieve the objectives of this research. For example, ovaprim as an exogenous hormone is widely accepted commercially in the aquaculture industry nowadays. Ovaprim as an exogenous synthetic hormone will be used for spawning induction to achieve synchronization and short duration of spawning under laboratory conditions. Ovaprim is a commercial preparation of salmon gonadotrophin releasing hormone (sgnrh). However, the optimum dose of ovaprim currently to induce spawning has not been elucidated in fish including tilapia. Therefore, this research was intended to evaluate the efficacy of ovaprim in spawning induction in tilapia fish. The success of this project will act as a model which will be later used for other important tropical fishes. 1

2 After a significant dosage of ovaprim is found to positively stimulate spawning, the next factor would be to ensure good quality and quantity of eggs are produced in order to achieve maximum hatching and survival rates under artificial incubation. Therefore, another aim of this study was to develop hatchery facilities in order to produce high numbers of fry under good supervision, for example, maintenance of good water quality is a critical checkpoint to achieve successful hatchery operation. Hatchery design is the most important step in getting the hatching process running in order. This would setup the dynamics of an artificial egg incubator for the success of the artificial incubation. Endocrine control of the reproductive cycle in fish including red tilapia is important. By understanding the physiological role of the reproductive hormone, for example, oestradiol, on spawning would enlighten the relationship between the hormonal responses in regards to spawning and related internal and external factors. Therefore, another objective of this study was to characterize oestradiol levels in the serum with reference to egg production parameters as related to body weight and body length. In our laboratory, radioimmunoassay (RIA) protocol was used to analyze the oestradiol levels in the serum of red tilapia. The final goal of this study was to produce good quality and higher quantity of viable eggs after the optimal dosage of ovaprim in an effort to obtain high hatchability and high number of fry survival. Practically, better production of juvenile tilapia means the fish stocks would be increased and consequently would not only economically enhance the local red tilapia industry but would also present opportunity for international markets. 2

3 Therefore, the main objectives of this study were: a) To develop a spawning protocol using ovaprim in red tilapia. b) To determine the effects of body weight and body length of red tilapia on egg production parameters, hatching rate and fry survival rate with reference to ovaprim stimulation. c) To determine the relationship of body size and oestradiol (E 2 ) levels after ovaprim stimulation with egg production parameters, hatching rate and fry survival rate. d) To compare between maternal incubation and artificial incubation with respect to hatching rate and fry survival rate after ovaprim induction. 3

4 Chapter REVIEW OF LITERATURE 2.1 REPRODUCTIVE BACKGROUND OF TILAPIA In the following sub-sections, historical background and research studies of tilapia reproduction will be described. These include sexual characteristics, fecundity, eggs, embryonic development and larval development Sexual Characteristics During spawning, the colouring of the male is more marked than for the female. The external differences between the sexes are not very clear. The external differences between the sexes are based on the fact that, the male has two orifices under its belly, in which, one is the anus and the other the urogenital aperture. The female has three; the anus, the genital and the urinary apertures. The anus is easily recognized. It is a round hole. The urogenital aperture of the male is a small point. The urinary orifice of the female is microscopic and is scarcely visible to the naked eye, while the genital orifice is an opening in a line perpendicular with the axis of the body. This transversal opening is situated between the anus and the urinary orifice (Beveridge and McAndrew, 2000). 4

5 Figure 2.1: Typical genital papilla of the tilapia (Masser, 1999) Fecundity Webster (2006) reported that fecundity can be expressed as the number of mature ova in the ovary, the number of ovulated eggs or the number of eggs deposited during spawning. These numbers may differ for tilapia fish of equal size. It means, not all mature eggs will be ovulated and not all ovulated eggs will be deposited during spawning. The number of ripe gonadal ova in tilapia species ranges from 1000 to One example is the fecundity of Mozambique tilapia based on mature eggs that range from approximately 100 to 1700 for females of 12 to 30 cm total body length. Macintosh and Little (1995) reported that an 8 cm body length Mozambique tilapia produces 100 to 150 eggs per spawning early in maturity and that the number increases with successive spawning and are able to produce more than 1000 eggs per spawning (Brummett, 1995). 5

6 The relationship between offspring numbers and fecundity is critical in terms of overpopulation and seedstock production. Green (1997) calculated that Mozambique tilapia in Lake Moyua, Nicaragua, produced between 2000 and 5000 eggs per year in 5 to 6 spawnings and that the number of recruits per female per season was 300 to Eggs The egg of tilapia is surrounded by several membranes, the nomenclature of which is not uniform (chorion, follicular epithelium, zona radiate, zona pellucida and vitelline membrane). These membranes in various teleosts undergo differential swelling after fertilization in relation to a particular reproductive guild (demersal and adhesive, demersal and nonadhesive, semibuoyant or pelagic). All species of tilapia have demersal eggs. Fertilized eggs are typically ellipsoid or ovoid and turgid from water infusion (water hardened) and cytoplasmic reorganization (Beveridge and McAndrew, 2000). Coward and Bromage (1999a) observed eggs to be yellow in colour, pear shaped and 2.0 mm by 1.75 mm in size. Chen et al. (1976) stated that eggs are elliptical with a long axis of approximately 2.5 mm. Eggs of O.aureus and O.niloticus are oval and orangeyellow in colour and they also range in size from 1.94 to 2.95 mm (McConnell, 1958). Generally, egg size of tilapia is species specific, although age or size of the female and nutritional state can influence development (Rana, 1988). Older female Nile tilapia produces larger eggs, which in turn results in larger fry. The volume of eggs range from approximately 2.8 to 11.1 mm 3 while 1-year-old females produce eggs of 1.9 to 2.8 mg mean weight, and 2-year-old females produce eggs of up to 3.7 mg mean weight (Macintosh and Little, 1995). Development of the eggs in the ovary goes through several stages and reaches the resting stage and may remain in this state for several months. This is a dormant egg, with a 6

7 micropyle and a central nucleus. In favourable conditions the dormant egg will start developing again until final ripening or ovulation and spawning. In the absence of favourable conditions, dormant eggs will degenerate and be reabsorbed by the ovary. The presence of favourable spawning conditions stimulates the further development of the dormant eggs in the ovaries. When environmental conditions become suitable, the brain signals the hypophysis to release gonadotrophins into the blood stream. When these hormones reach the ovaries, they trigger the final ripening of the dormant eggs and spawning. Males also become ready for spawning on gonadotrophin command (Masser, 1999). The maturation of the eggs will start when the central nucleus migrates toward the micropyle, and hydration slightly increases the egg size. Then, ovulation occurs during spawning in which the membrane of the nucleus disappears and the chromosomes become visible, the first cell division takes place, the egg follicle dissolves liberating the egg from the ovary wall, and the ripe egg is spawned (Shelton, 2002). The number of eggs varies according to the species and the size of the brood fish. There are, for each spawning, several hundred at least and several thousand at most. Nile tilapias (75 to 500 g) deposit from 50 to 2,000 eggs per spawn (Rana and Macintosh, 1988). Finally, it is possible on count to several hundred fry. After the eggs hatch, the female again leaves the school for whatever cover is available in shallow water areas (Turner, 2000). Eggs hatch in 2 to 3 days. Fry remain in the female s mouth until yolk sac absorption, 5 to 8 days later, by which time the swim bladder has developed, permitting the fry to swim well. Nevertheless, they often seek refuge in the female s mouth at the slightest sign of danger. As the fry grow they disperse. Gradually, the school of fry breaks up and at the end of 10 days it is practically disintegrated. The fry now swim around in shoals. 7

8 2.1.4 Embryonic Development According to Rana and Macintosh (1988), incubation time for Nile tilapia is inversely and linearly related to temperature. For example, time to hatching varies from approximately 2 to 3 days at 34ºC, however, hatching can be delayed to 8 days at 17ºC. Galman and Avtalion (1980) described that development of embryogeny occurs at 27ºC and by Rana (1988) at 28ºC. After a sperm penetrates the micropyle, cellular reorganization ensures the formation of a cytoplasmic cap beneath the micropyle on the yolk surface. This process stimulates the resumption of chromosomal redistribution during the second meiotic division and the formation of the second polar body. Subsequently, the newly formed zygotic nucleus initiates the first meiotic division (karyokinesis), which is followed by cleavage furrow development (cytokinesis) to form the 2-cell zygote (Chhorn, 2006). The fertilized eggs will cleave and initiate gastrulation after approximately 12 to 15 hours at 27ºC (Galman and Avtalion, 1980) or approximately 10 to 12 hours at 28ºC (Rana, 1988). By 72 hours, the 3 main divisions of the brain, i.e. the optic buds, the otic capsule with otolith nuclei and a few somites, have formed while the tail is free from the yolk surface. Pigmentation, particularly in the eye and on the yolk surface is present after between 72 and 100 hours in normally pigmented fish in the red colour mutant of the Nile tilapia (Shelton, 2002). The embryo finally appears with tail, head and eyes. It develops into a larva and hatches, breaking out of the egg shell. The newly hatched larva is still very different from the adult tilapia because it has no mouth and is nourished from the yolk sac. After about 4 days at 20 to 24ºC, the mouth is formed, the swim bladder is inflated with air and this is the beginning of the fry stage. In Mozambique and Nile tilapia, eggs will hatch in 3 to 5 days 8

9 and the total time from spawning until the young are no longer taken into the mouth is approximately 10 to 14 days (Chhorn, 2006) Larval Development At the first stage of larval development, the pigmented eyes are very prominent under gross observations. There are some chromatophores present on the surface of the yolk at hatching. The process of yolk absorption and swim bladder development permit buoyancy control and at the same time the mouth develops approximately 4 to 5 days after hatching. According to Webster (2006), the incubation and brooding period for eggs and larvae of Mozambique tilapia kept in plastic pools was reported to be 20 to 22 days and newly hatched fry was measured at 5 mm. On the other hand, Rana (1988) mentioned that the size of Nile and Mozambique tilapia larvae is affected by the size of the egg relative to the amount of yolk stored. At 8 to 12 days, the larvae are approximately 8 mm and most of the yolk has been absorbed. The fry can swim with some buoyancy control because at that time the swim bladder is developed and the pharyngeal arches and jaw also have formed and are functional (Carral et al, 1992). Lorenzen (2000) found that, Mozambique fry at 26ºC began to feed on the seventh day after hatching, even though a considerable amount of yolk remained. At that time some cannibalism in young under culture conditions might happen and the survival of fry until transition to active feeding was 93%. In conclusion, the relative fecundity based on Little and Hulatas (2000) data, reported survival rate from fertilized egg (in the mouth) to become fry at 36%. The larvae are usually reared in larger aquariums with fine supply of oxygenated bubbles until they develop into early fry, which feed on exogenous food. It is very important not to forget to check that the water temperature in the various containers should 9

10 be similar. As soon as the early fry accept exogenous food, they should be removed from the rearing aquariums and stocked in rearing tanks. In addition to the natural food which is important to early fry, it is necessary to provide artificial food to ensure the best possible growth and survival during this first month of rearing. The artificial food consists of a mixture of equal parts of soybean meal (1), wheat meal (2), fish meal (3) and blood (or meat) meal (4). It is in the form of a very fine dry powder made of 0.1 to 0.2 mm particles (Chhorn, 2006). 2.2 HORMONAL MANIPULATION IN FISH SPAWNING INDUCTION During the past two decades, induced breeding by carp pituitary extract has been attempted in ornamental fishes (Zohar and Mylonas, 2001). As times go by, increasing cost of donor pituitary and the cumbersome process have initiated experts to test alternative hormones such as human chorionic gonadotrophin (hcg) (Zairin et al., 1992), luteinizing hormone releasing hormone (LHRH) (Hassin et al., 1998) and ovaprim (Watanabe et al., 1994). The present study involves the use of ovaprim on induced breeding of Oreochromis niloticus from the commercial aquaculture species in Malaysia. Ovaprim (Syndel Laboratories, Ltd.) is a mixture of salmon gonadotrophinreleasing hormone analogue (sgnrha [D-Arg6-Pro9-Net sgnrh]) with dopamine antagonist, domperidone (Head et al., 1994). Ovaprim is a common spawning inducer in numerous fish species in aquaculture except lack of its applications on the red tilapia, Oreochromis niloticus. Therefore, the red tilapia may be considered as a model species for modifying spawning induction techniques. From ovaprim injection, we should be able to predict how the reproductive traits of Oreochromis niloticus will improve in terms of spawning time, spawning frequency, number of eggs produced, hatchability and survival of fry. 10

11 In 2002, Haniffa from the Center for Aquaculture Research in South India was successful in carrying out a spawning induction using ovaprim and human chorionic gonadotrophin (hcg) by administering a single intramuscular injection of the hormones to the spotted murrel (Channa punctatus) at varying dosages. It was observed that fecundity in C. punctatus was 3273±75 eggs for ovaprim and 1253±126 eggs for hcg. Successful spawning of C. punctatus was observed at 0.3 and 0.5 ml/kg body mass for ovaprim and at 2000 and 3000 IU/kg body mass for hcg. They found that an increase in dosage of ovaprim and hcg elevated egg output where the enhancement was statistically significant (p<0.05). Besides that, no spawning was noticed in low dosages of both ovaprim (0.1 ml/kg) and hcg (1000 IU), which indicated that the selected dosages were insufficient to induce spawning (Haniffa, 2002). Previous authors found that the dosage of ovaprim for induced spawning in carp and murrel ranged between 0.3 ml/kg and 0.6 ml/kg body mass (Haniffa, 1998). Haniffa (1998) also reported that a fertilization rate for C. punctatus injected with ovaprim was the highest ranging from 95% to 98%. Then, this was followed by LHRHa (75% to 80%), hcg (65% to 79%) and pituitary extract (60% to 70%). This showed that ovaprim reported better performance in inducing higher spawning frequency, fecundity and viability of the eggs. Different dosages of hormone mostly used in spawning induction may affect fertilization and hatching rate in certain teleosts. According to Lee et al. (2001), induction of oocyte maturation in cultured female Korean spotted sea bass (Lateolabrax maculates), showed that in the case of 1000 IU/kg of hcg injection, the fertilization rate and hatching rate were 75.7±1.8% and 72.7±7.5%, respectively. While in the case of 2000 IU/kg of hcg injection, the fertilization and hatching rate were 70.1±5.0% and 65.8±5.9%, respectively. They concluded that finding a potent dosage in spawning induction for specific species is detrimental for obtaining optimum results in egg production and viability. 11

12 In addition to dosage and types of spawning induction hormones, administration of the hormone also may affect the reproductive performance in certain species. Lee et al. (1998) emphasized that, egg quality depends on the particular hormone used or type of administration protocol. In 2005, Watson from the Tropical Aquaculture Laboratory, USA, found that intramuscular injection (IM) of ovaprim produced the highest ovulation (100%) in induced spawning of a tropical ornamental fish compared with topical gill application of dimethyl sulfoxide (DMSO) and ovaprim (78%), however, topical gill application of DMSO alone produced no ovulation of females. In this case, route of hormone administration is very important for giving positive hormonal action physiologically on the target organ especially in stimulating egg production or ovulation. 2.3 ARTIFICIAL EGG INCUBATION With regard to the reproductive phase, many authors have reported that high egg or embryo mortalities while they are incubated to the female throughout the embryonic development (maternal incubation). Mortalities might occur for various reasons, for example, cannibalism from parents (Rana, 1988), egg-bearing females dying (Arrignon, 1981) and excessive handling of mouth-brooding females under culture conditions may contribute towards egg losses (Tyler and Sumpter, 1996). Carral et al. (1992) from the University of Leon in Spain minimized this problem by developing artificial incubation techniques for freshwater crayfish eggs. This research can be a good concept for reproductive performance in tilapia fish. They made comparisons between maternal and artificial incubation and demonstrated the suitability of the tested system for artificial incubation for eggs of this species. The results showed that overall survival rate to juvenile was significantly higher (p<0.05) in artificially incubated eggs (68.2%) compared with those attached to the maternal pleopods throughout the embryonic 12

13 development (56.2%). It can be seen that artificial incubation provides at least similar results to those of maternal incubation. Further analysis on achievement between maternal and artificial incubation, hatchery production of Florida red tilapia seed in tanks was studied on Lee Stocking Island (Exuma Cays, Bahamas) under two methods of broodstock management. Seed production over a 3-month period under the natural-mouth-brooding method (3.3 seed m 2 day 1 ) was significantly lower (P<0.001) than that obtained under the clutch-removal method (91.7 seed m 2 day 1 ) which is among the highest reported for tilapia hatcheries. They concluded that poor seed production under the natural-mouth-brooding method was attributable to cannibalism of egg and fry by adults (Zohar and Mylonas, 2001). There are many significant factors that must be pointed out in artificial egg incubation. One of the significant factors that affect egg incubation is temperature. Perez et al. (1999) stated that, yolk sac absorption was faster at higher incubation temperature (33 C). From their study, the optimal temperature for incubating Labeo rohita eggs was recorded at 31 C taking into consideration the rate of development and hatching percentage, which was higher than earlier reports on Labeo rohita. Various studies have reported that, embryonic development in fishes is dependent on many factors in addition to light, ph and dissolved oxygen (Bromage et al., 1991). Craik (1985) reported an investigation on sea trout, brown trout and rainbow trout in which they were kept in three types of lighting conditions. After carrying out experiments on hatching apparatus in wooden hatcheries and ponds over 5 years, the following conclusions were made: firstly, the intensity exceeding 330 lux showed negative effect of light on the egg incubation of rainbow trout; secondly, increase in mortality following the moment of eggs eyeing compared with the eggs not exposed to light; thirdly, a confirmed worsened growth of trout larvae maintained in complete darkness and finally, 13

14 no changes in pigmentation of rainbow trout kept in continuous artificial illumination and continuous complete darkness were observed. The most important success in artificial incubation is dependent on the artificial incubator itself. If the incubator has reached suitable dynamic conditions similar to the maternal incubation, it will give high production rate in any hatchery production. In Florida red tilapia, hatching success of eggs in upwelling incubators averaged 64.8% with effects of densities of 462, 923, 1385 and 1846/L not differing significantly (P>0.05). Fry obtained from artificial incubation were significantly (P<0.05) larger (9.9 mg vs. 7.5 mg) and more viable than those obtained through natural mouth-brooding as evidenced by higher fry survival rate (73.9% vs. 49.7%). The results suggest that the clutch-removal method yields markedly higher numbers of viable fry for grow-out (Liao, 1993). In every hatchery operation, they will do all the routine collection and handling practices including pouring of eggs from the collectors to pails and to incubation tanks through a sieve, siphoning of eggs, lifting or dispensing eggs with nets and volumetric measuring devices and transporting eggs to hatchery facilities. During these manipulations, fish eggs are exposed to varying degrees of mechanical shock. According to Garcia (1997), the variability in shock sensitivity has been speculated to be related to interspecies and developmental differences in egg property and structure. To avoid unnecessary mortalities and the possible occurrence of larval deformities, sensitive stages of all egg species to physical stress must be determined. In many recent studies, natural or induced spawning dominates the fish hatchery industry, as it is more effective than the strip method (Liao, 1993). It is time-consuming and relatively ineffective. Mortality of spawners often occurs, particularly with the large black carp. Similarly, Juario (1984) reported that the resultant fertilization of the strip method in milkfish is relatively low. It has also been demonstrated by Harel et al. (1992) 14

15 that a lower fertilization rate was obtained with hand-stripped batches of Atlantic tomcod (Microgadus tomcod). Liao (1993) pointed out that induced spawning involved the use of a well-balanced and sound approach to hatchery practice. It has several advantages, such as spawner-saving, water-saving with high fertilization and hatching rates. Kuo et al. (1995) also concluded that the induced spawning method has a consistently high fertilization rate and, more importantly, reduces damage to broodstock β-OESTRADIOL MEASUREMENT IN INDUCED SPAWNING FISH The association of external spawning inducing hormone administration with plasma levels of gonadal steroids has proven to be a valuable tool in the development of an understanding of endocrine control of reproduction in teleosts. With the use of the radioimmunoassay (RIA) technique, the study of changes of plasma oestradiol levels in fish during spawning induced by 1.0 ml/kg ovaprim were carried out, which indicated the contribution of synthesis and release of oestradiol to the modulation and control of spawning. In other words, RIA could be developed for measuring the oestradiol in fish plasma and subsequently laying out a foundation for research on the function of sexual hormone in fish. It is well known that in teleosts, vitellogenesis and final oocyte maturation are regulated by gonadotrophins via steroid secreted by the follicular cells surrounding the oocyte. Of these steroids, oestradiol stimulates hepatic synthesis and secretion of vitellogenin which is accumulated in the oocytes (Kobayashi et al., 1996). Correlations between changes in plasma levels of gonadal steroids and oocyte development have been well documented in a number of freshwater species including salmoniforms, cyprinids, catfish (Heteropneustes fossilis), goldeye (Hiodon alosoides) and walleye (Stizostedion vitrum) (Lee et al., 2001). 15

16 For example, in normal reproduction of marine fish, Lee et al. (2001) discovered the relationship between ovarian development and plasma levels of gonadal steroid hormones in Korean spotted sea bass (Lateolabrax maculates) to show that changes in plasma levels of gonadal steroids were correlated with ovarian development. Plasma oestradiol level were 90.3±15.4 pg/ml in August and increased in September (190.5±21.4 pg/ml), reaching their highest levels in October and early November (394.6±75.3 and 380.2±96.6 pg/ml). Then the levels decreased to 202.7±42.1 pg/ml in mid-november. The highest GSI value and the decrease in plasma oestradiol in mid-november indicated that the ovaries were filled with post-vitellogenic oocytes. In many cases, captive females failed to not only spawn but also to complete vitellogenesis and oocyte maturation (Bruce et al., 1999). In addition, maturation-inducing steroid (MIS) was not produced in many species including catfish (Clarias macrocephalus) and white bass (Morone chrysops) (Mylonas et al., 1997). Therefore, induction of maturation and ovulation by hormone treatment especially ovaprim induction must be conducted in order to solve any spawning problems that will be done in laboratory conditions. Based on observations done by Lee et al. (2001), the levels of 17β-oestradiol in the presence of hcg (410±30 pg/ml) were much higher than those of control (80±10 pg/ml). These results suggest that one reason cultured fish fail to mature may be due to lack of a gonadotrophin surge. The lack of gonadotrophin surge in the cultured fish is probably due to the dissimilarity in environmental conditions between the culture system and the natural environment (Donaldson and Hunter, 1983). Although it has been ascertained in cyprinids that final oocyte maturation and ovulation are induced by a preovulatory gonadotrophin surge, little information on the plasma and gonadal changes in oestradiol levels during the induced reproduction in 16

17 O.niloticus is known. Smith and Haley (1988) reported that working on female O. mossambicus, there is no decline in oestradiol levels prior to oocyte maturation. The results may imply a close interaction between ovaprim stimulation and endocrine control of reproduction. Naturally, endocrine control cannot continue without the appropriate environmental cues required to stimulate reproduction. This may be used as a starting point for breeding in controlled laboratory conditions when attempting to manipulate artificial breeding by hormonal intervention. 2.5 METHODOLOGY The review of the methodology section includes broodstock selection, application of ovaprim, hormonal injection techniques, spawning and quality of eggs, hormone study through radioimmunoassay techniques, incubation and hatching and larval rearing Broodstock Selection Broodstock management covers three particular aspects of the rearing process. The selection of fish with desirable hereditary qualities typical of improved strains such as rapid growth potential, higher resistance to dissolved oxygen deficiency and adverse water quality and strong appetite. The selection of fish with well-developed sexual organs is a very important step. The rearing of these selected fish to produce healthy potential spawners, with dormant eggs well developed in the females. The selection of good future tilapia breeders should take into account the general shape of the fish body, scale distribution, state of health and development of sexual organs. In particular, the selected fish should be in good health, with no body wounds, no parasites, a typical scale distribution and no fin or body deformation (Zohar and Mylonas, 2001). 17

18 The body should possess the required shape and proportions. Male and female breeders may be easily differentiated by the shape of the body and the relative position of the genital papilla. In females, the genital opening is situated above the genital papilla. In males, the genital opening is found behind the genital papilla. The differences between male and female fish can be compared in the illustration in Figure 2.2 which shows the external appearances of the sex organs of mature tilapia. To check whether a tilapia breeder has reached maturity (presence of dormant eggs or sperm) and may be selected for artificial propagation, genital papilla should be examined carefully in order to know their readiness to spawn. A mature female has reddish genital papilla; its vagina opening is clearly seen by naked eyes (Chhorn, 2006). Figure 2.2: Features to differentiate between male and female tilapia fish (Beveridge and McAndrew, 2000). 18

19 Female breeders are then categorized in one of the following maturity conditions: ready to spawn, swollen, not ready to spawn and has spawned. Descriptions of these categories are given in Table 3.1. Female breeders that are categorized as ready to spawn are first selected for pairing with males. A mature male will release milt under slight abdominal pressure. This selection process is especially important for the females, whose maturity should be thoroughly checked to ensure the success of the artificial propagation. It is important to avoid dissolved oxygen deficiency which may damage the sensitive breeders during seining and selection. During handling, fresh water may be pumped into the crowded enclosure if necessary Application of Ovaprim Hormone injection is used to induce spawning in numerous fish species in aquaculture (Zohar and Mylonas, 2001). The process of final maturation (migration of the nucleus to the animal pole, fusion of the yolk, breakdown of the germinal vesicle followed by first meiotic division) and ovulation (rupture of the follicles and accumulation of the ripe eggs in the ovary cavity) cannot be stopped or reversed after administration of the correct hormone dosage. Once these processes start, the eggs must either be spawned or stripped (Sarma, 1994). Injections of gonadotrophin hormones induce the final ripening of the dormant eggs in the selected females. These injections replace the natural release in the bloodstream of such hormones by the hypophysis, at the command of the hypothalamus. Only two basic environmental factors have to be at optimum level: water temperature and dissolved oxygen content. The dose of hormones to be injected to the spawners is based on the live weight of each spawner. As far as possible, it is better to use batches of spawners similar in size to simplify calculations (Sridhar and Haniffa, 2002). 19

20 Ovaprim was concurrently used to induce final maturation and spawning in freshwater fishes. From the data of trials, ovaprim inducing fish spawning is better than the products used before and it can also induce final maturation, increase milt production and improve the fecundity greatly (Nandeesha et al., 1990). Ovaprim is the product of Freshwater Fisheries Research Center (FFRC) cooperated with Syndel International Inc. Canada on the experiment for fish hormone product. FFRC introduced the Syndel ovaprim product in April Experimental targets in their trial involve response time (time from treatment to spawning), spawning rate, fertilization rate, hatching rate and survival rate (Little and Dawson, 2002). Ovaprim contains Syndel s patented analogue of salmon GnRH (SGnRHa) and domperidone, a dopamine inhibitor, in a sterile liquid for easy injection. It is used for injection directly and easily because it needs not to be dissolved or prepared. SGnRHa works with the natural spawning system of the fish. When injected, it causes the release of natural hormones in the fish brain and gonads to trigger normal spawning and spermiation. Domperidone blocks the negative effects of dopamine that normally inhibits hormone release. In hard to spawn fish, domperidone is essential to support the effectiveness of SGnRHa. Ovaprim must also be used during, or at the start of the spawning season (Sridhar and Haniffa, 2002). In general, the reproductive processes of fish are regulated under the control of the hypothalamo-pituitary-gonadal axis. Within this axis, gonadotrophin-releasing hormone (GnRH) secreted from hypothalamus of the brain play an important role in the pituitary gland. GnRH stimulates gonadotrophin (GTH) release in the pituitary gland and the GTH activates the maturation competence of oocytes via synthesis and release of the maturationinducing steroid in the ovary and these hormones induce final maturation and ovulation (Peter et al., 1988). 20

21 The proper dosage of ovaprim must be calculated for the brood fish and the optimum injection schedule must be used for best results. To calculate the proper dosage, (a) the recommended dose, (b) approximate weight of the brood fish and (c) the desired volume of the injection must be determined. The quantity of ovaprim to be injected can then be calculated from the weight of each individual or groups of brood fish and the appropriate injection scheduled. The formula for the calculation of ovaprim amount to be injected is stated as below which has been taken from the Syndel Laboratory (2003) protocol: Weight of fish X Ovaprim dosage = Amount of injection This dosage varies among species and locations. Ovaprim has been successfully tested on Thai carp Puntius gonionotus in Thailand, walking catfish Clarias batrachus in Malaysia and in Australian eel-tailed catfish (Shivpuri, 2000) Hormonal Injection Techniques The acute injection or infusion of a variety of substances into fish is a common and useful procedure in physiological studies. The list of such substances frequently injected into fish is exhaustive including hormones, metabolites, radio labeled compounds and enumerable bioactive drugs. The method and route of administration of the specific compound may vary according to its chemical properties, target site, rate of degradation and the required duration of treatment. Acute administration is where the substances to be injected are generally dissolved or diluted in the appropriate medium termed the vehicle. Importantly, the chemical composition of the vehicle, whenever possible, should resemble the chemical composition of fish extracellular fluid. This is easily achieved by using physiological saline as the 21

22 vehicle. This is referred to as basic recipes for the preparation of physiological salines for freshwater and seawater teleosts. The ph of the vehicle usually is adjusted between 7.8 to 8.0 by using HCl or NaOH (Sarma, 1994). The site and method of injection is dependent upon the desired duration of the treatment period, the chemical properties of the substance and the nature of the particular study. Common methods include intravascular injection, intraperitoneal injection, intramuscular injection, oral administration, emersion and implantation of osmotic pumps, silastic pellets or oil pellets (Zairin et al., 1992). Among these methods, direct injection into the circulation is a convenient and reliable technique for rapid delivery of compounds. In many teleosts, the site of injection or blind puncture is the caudal vein or caudal artery. There are two common places to inject hormones into a fish. An intraperitoneal (IP) (within the body cavity) injection is given through the ventral (bottom) part of the fish behind either the pelvic or pectoral fin. Intramuscular (IM) (within the muscle) injections are commonly done on the dorsal (upper) part of the fish above the lateral line and below the anterior part of the dorsal fin. It is difficult, if not possible, to selectively inject into the caudal vein versus the caudal artery but one can nevertheless be sure that the substance will eventually be delivered to the entire circulation unless its biological half life is extremely short (less than 1 minute) (Little et al., 1987). While the caudal puncture is performed, the needle is advanced until there is contact of the tip with vertebra and then the compound is injected. It is important to place the needle so that it slides under the scale rather than through it, penetrate the body wall and slowly deliver the hormone into the body cavity. This work needs to be done in sterile equipment in order to limit infection potential. Before withdrawing the needle, leave it in place for a few seconds after delivery of the hormone because this will prevent back- 22

23 seepage. Commonly the wound bleeds for several minutes after the needle is removed but the blood loss is normally not substantial. The blind puncture technique is used frequently to inject the substance to in-situ tissue (Watanabe et al., 1994). Many bioactive compounds can be toxic or produce adverse side effects when injected too rapidly at high concentrations. Thus, it is often advisable to infuse the compound slowly (e.g., 1.0 ml/h) into the cannula using a syringe pump. Intravascular infusion is an accepted method for long term delivery of compounds into the circulation. In such cases, the rate of infusion must be kept reasonably low so as to avoid volume loading and the long term stability of the compound being infused must be assessed. Although injected compounds are initially mixed with the vascular fluid (about 3% to 5% of body mass), they rapidly equilibrate in the extracellular fluid compartment unless the compound is too large to be filtered across capillary endothelia. Thus, to accurately determine the quantity of substance to be injected, one should know the volume of the space that it will ultimately dissolve in. For the majority of compounds, this can be estimated as the extracellular fluid volume or about 30% of body mass. While the calculation of the delivered dose of a compound (e.g., mg/kg/h) is straight-forward, it is much more difficult to estimate the circulating levels in the blood based on the given dose. Ultimately, the circulating levels depend upon several factors of which the biological halflife of the compound is probably the most important. Furthermore, the half-life may vary during a prolonged period of infusion owing to changes in the metabolic clearance rate (Watanabe et al., 1994). Two dosage levels are commonly used: a preparatory dose and a decisive or final dose with a time gap generally of 12 to 24 hours between the two injections. The preparatory dose brings the fish to the readiness of spawning and the decisive dose induces ovulation. In general, the preparatory dose is about 20 percent of the total dose. These 23

24 injections are useful if slow release of a compound into the extracellular fluids is desired. These procedures impart considerable stress to the animal. Thus the fish must be restrained prior to injection. In order to simplify the handling and injection, the fish may be lightly anaesthetized (until the equilibrium is lost) using a solution of 1:10,000 (w/v) MS222 (Myers and Hershberger, 1991) Spawning and Quality of Eggs The eggs and milt of fish can be taken by several different methods: (a) tank spawning (b) hand stripping, and (c) surgically removing eggs. The method of choice depends on the fish species, hatchery facilities, experience and skill of the hatchery staff and the desired manipulation of eggs, sperm or fertilized eggs. Because the internal anatomy of fish vary greatly, therefore hand stripping may be difficult in some species. The simplest method without any surgical method or killing them for obtaining eggs is by tank spawning. The females will ovulate when hormonal induction is done. The males stimulate the females to release the eggs and fertilize it. This method is very convenient and ready to apply in this study. Fertilization and hatching success are normally used as egg viability indicators but such parameters do not specify what components were responsible for egg viability (Brooks, 1997). The quality of the eggs also can be determined by the contribution of the paternal genes. Both in aquaculture and in nature, the environment in which the eggs are incubated also affects the success of the egg in producing a viable offspring. Egg quality is currently defined as the potential of an egg to hatch into a viable larva. The good quality of eggs is also characterized by increased translucency and, however, an aggregation of cytoplasm at the animal pole will reduce fertilization and 24

25 hatching (Lam, 1978). Specific components that determine egg quality and methods in assessing them have not been established especially in many cultured tilapia. The low quality eggs are dark and clump together (Shireman, 1991). Poor hatching of eggs may be due to the manual expression of eggs before final maturation of the ova, hence fertilization ability was reached (Saat, 1993). Indeed, the fish were not heavily conditioned, and the eggs were not staged prior to the spawning attempt. Garcia (1997) has also reported poor egg quality with increasing doses of gonadotrophin. It is usual that higher doses results in early ovulation and the ovulated eggs remaining in the ovarian lumen in hypoxic conditions for a longer time can lead to over ripeness (Hill, 2005). In terms of low fertilization, this might be due to asynchrony between maturation and ovulation that can lead to low hatching and is in agreement with the report of Shelton (1999). More deformity in larvae at lower or higher dose may be attributed to the fertilization of unripe or over ripe ova. Lam, (1978) noted that over ripe eggs did not form a perivitelline space when placed into fresh water, suggesting that there had been a change in the permeability of the chorion. Consequently reduced permeability of the chorion to water may adversely affect utilization of the yolk, leading to retarded or abnormal embryonic development in the over ripe eggs (Smith, 1957). Goswami (1997) have reported similar highly deformed larval production in C. batrachus at lower and higher dose of pituitary respectively during induced breeding Hormone Study Through Radioimmunoassay Techniques The association of changes in gonad condition with plasma levels of gonadal steroids has proven to be a valuable tool in the development of an understanding of endocrine control of reproduction in tilapia. Correlations between ovaprim induction in plasma levels of gonadal 25

26 steroids and spawning were documented in a number of induced tilapia (Mylonas et al., 1997). In general, oestradiol is responsible for stimulating vitellogenesis and is also secreted by female gonads during the pre-spawning period (Smith and Haley, 1988). During vitellogenesis, an increase in plasma oestrogen levels, mainly 17β-oestradiol that correlates with the growth of vitellogenic oocytes has been observed in many species. Evaluation of the plasma steroids especially oestrogen reflects the importance of this hormone. Studies on the estrogenic effects in fish are based on the administration of estrogens, usually in high doses. In order to clarify the fate of exogenous oestradiol in tilapias, ovariectomized females were injected with an exceedingly high dose of steroid (0.5 mg/kg) dissolved in sesame oil, and their plasma was sampled at intervals. The circulating level of estrogen five hours after injection was extremely high. Then, a physiological level about 1-10 ng/ml was reached after 24 hour and was maintained in the subsequent 4 days (Yaron et al., 1978) Incubation and Hatching The importance of a constant supply of quality fry in tilapia culture has generated considerable interest in the hatchery rearing of tilapia eggs and early fry. This has led to experiments on the development of various types of artificial systems which simulate special conditions characterizing oral incubation. Related to this experiment, artificial incubation systems for mouth-brooding tilapias need to be designed to minimize mechanical stress to the eggs, which is very detrimental to their survival (Sommerville, 1985). Some authors have considered the possibility that artificial incubation may equal and even improve the results obtained from maternal incubation under culture conditions. 26

27 This has been supported by the results of some experimental studies, where maternal and artificial incubation were compared (Evans et al., 1993). Yang and Kwak (2004) reported that seed production over a 3-month period under the natural mouth-brooding method (3.3 seed per square meter per day) was markedly lower than under the clutch-removal method (91.7 seed per square meter per day), which is among the highest reported for tilapia hatcheries. Poor seed production under the natural mouth-brooding method was attributable to cannibalism of eggs and fry by adults. Although of similar ages, post-yolk sac stage fry obtained from artificial incubation of eggs and yolk sac fry were larger and more robust than naturally incubated fry, exhibiting higher survival (73.9% vs. 49.7%) over a 29-day culture period (Watanabe and Kuo, 1992). This is partly attributable to the availability of prepared feeds to artificially incubated fry at first feeding, whereas naturally incubated fry are dependent upon foods available in brood-fish tanks (Rana, 1986). In Oreochromis spp., a delay in initial feeding depressed growth rate of swim-up fry and reduced viability. Early availability of prepared feeds may have also improved survival among artificially incubated fry by reducing cannibalism. Therefore, greater size-age uniformity among artificially incubated fry may have also minimized aggressive interactions and cannibalism (Rana, 1988). As the embryo grows, it starts to move as well. The first thing that is usually seen moving on an embryo is the heart and more accurately the heart sac. This heart sac starts to contract. Then slowly it starts to move other parts of the body. Twitches are evident throughout the embryo. After the twitching of the body itself the tiny developing pelvic and pectoral fins begin twitching. The fanning motion of the fins is very important to the development of the embryo (Chhorn, 2006). 27

28 The hatching process is a result of a variety of activities that the embryo is going through. In addition to these activities the chemical and enzymatic readiness of the embryo to hatch is crucial to a successful hatch. The embryo grows to such an extent that it needs more and more oxygen until finally it becomes desperate in its oxygen use. Through the increased need for oxygen, it starts to develop hatching enzyme glands. These glands are spread over the whole body but are concentrated primarily on the head and near the operculae (gill covers) on the side of the head. The hatching glands are triggered to release their contents when the oxygen shortage becomes acute (Brooks, 1997). The glands spread their contents through the vitelline fluid that surrounds the embryo. The embryo assists this essential spreading of fluids by the fanning motion of the developing fins. The fanning produces the right mix of enzymes throughout the entire body of the embryo. The enzymes within the zona radiata (capsule) gradually weaken the membrane. At the same time, the embryo is struggling for oxygen and finds the egg shell limiting. When the embryo increases its stretching and pushing movements it will burst the weakened membrane. An embryo which has not been actively wriggling and squirming, because of a number of genetic or environmental reasons, will have released the hatching enzyme but the enzyme will not have been distributed throughout the body. The enzyme will have remained concentrated locally around the region of the head. These embryos may attempt to push and stretch but only their head will break through the shell. Since they are incapable of wriggling out of the capsule, they will die because only their heads are out of the capsule (Abdelghany, 2000) Artificial egg incubator Most teleosts species require that their eggs incubate and hatch in open water. Eggs are broadcast in the water column and either float or sink; adhesive eggs may attach to plants or 28

29 hard substrates such as wood, rocks or gravels. Some fish eggs are laid in a nest and the parent provides a constant water flow by fanning their fins in order to give aeration to the eggs. Some fish also incubate eggs in their mouths where movement of the gill plates provide both gentle tumbling and water circulation like red tilapia. Artificial incubation and hatching of fish eggs simulate these natural processes. In the wild, eggs are susceptible to predation and also are easily damaged by the continual change of the natural environment (Beveridge and McAndrew, 2000). The developing embryos and newly-hatched larvae are the most sensitive and delicate of the stages in the life history of a fish. Therefore, great care must be taken to provide them with the proper incubation and hatching environment. The advantage of this man-made hatchery is that the environment can be controlled and manipulated. Water temperature, light, water quality, water flow, shock prevention and types or sizes of the eggs are very important considerations. Hence, artificial incubation of fish eggs is a hatchery practice that will increase the economic efficiency of commercial fish culture operations. For many years, the industry depended on fingerlings produced through daily scooping of fry in the breeding pond. The unexpected problems caused by this practice included poor growth and deformities due to inbreeding, the poor production of fry caused by cannibalism in the breeding pond and irregular sizes due to uncontrolled breeding regime and other factors. Thus, an artificial egg incubator system must be developed to support the thriving and expanding tilapia industry especially for small or individual breeders. It is aimed to help hatchery operators produce healthy fry of desired size, strains and quality. This could support either backyard type or upgraded conventional scale hatcheries (Rana, 1992). Due to increasing demand of tilapia fingerlings nowadays for fish farmers or villagers, the artificial egg incubator system is practiced to speed up the hatching of eggs 29

30 and shorten the breeding cycle of tilapia in captivity. The adoption of this simple and safe system has proven that it is a worthy fingerlings production. It has now attained higher fry to fingerling production at better quality than traditional practices. The artificial egg incubating system can be used for incubating eggs of either tropical or marine fish whether it is induced or naturally spawned. Because of flexibility in material components and design of this system, it is adoptable by the majority of fish farmers especially whose breeding tilapia in their homes. This system can be designed on a big scale for massive fry production, like commercialization of fisheries agencies. Likewise, this can be a tool for promoting a sustainable aquaculture system. In conclusion, artificial incubation of fish eggs is a hatchery practice that will increase the economic efficiency of the commercial fish culture operation. Hatching rates and survival rates could be increased using artificial incubation. Removal of the eggs from the parents also may increase egg production by shortening the time for another spawning to occur Types of egg incubator According to Frank (2002), there is a wide variety of devices used for incubating fish eggs. For practical purposes, there are three major types of fish egg incubators: egg mats, trays and conical incubators. Their usage is based on the density of the eggs to be hatched, their stickiness and sensitivity of the eggs to mechanical shock. For adhesive eggs, egg mats are used by simulating a spawning substrate (rocks, wood, etc.) and they serve as egg collectors and provide a place for egg attachment. Egg mats also serve as a stimulus for spawning and are known as spawning mats. Mats consist of bundles of fibrous material arranged in a variety of forms and made from a variety of different materials such as plastic shreds and coconut fibers. Typically, egg mats are suspended in the water column of the spawning 30

31 container. This type of incubator is used for spawning and incubating eggs of many ornamental fish such as angelfish, discus and catfish. A tray-type incubator consists of a container that is perforated, through which a flow of water permeates to supply the eggs with oxygen and flush away waste products. They are often designed so that water can penetrate the tray from below and flow out over the upper edge. Since the eggs lay over a screen, tray-type incubators are ideal for fish eggs that can be injured by movement during incubation. Tray incubators also can be stacked and provide easy access for removal of dead eggs or embryos. Tray type incubators can be formed into baskets and are commonly used to incubate and hatch channel catfish eggs. The baskets are placed in a water trough and paddle-wheels in order to provide aeration and gentle circulation of the water. Baskets can also be placed outside the spawning tank and then used as incubators. The hapa or net enclosures traditionally used for spawning, egg incubation and larval rearing of common carp function similarly to basket or tray incubators (Chhorn, 2006). Fish eggs that are non-adhesive and require constant movement are commonly incubated in conical shaped tanks or jars where water flows into the bottom or top of the container. In this type of incubator, the eggs are gently suspended and constantly tumbled in the lower portion of the jar. Well oxygenated water is constantly being replaced in the jar and it will keep the eggs from collecting debris which can lead to fungal infections. It is advantageous to use a screen because greater surface area is provided for water to flow out, preventing the egg, yolk-sac larvae or the larvae from becoming crushed. Incubators made of net material require structural support and must be suspended inside a larger tank or placed into the rearing tank (Brooks, 1997). Incubation and hatching containers are extremely cost effective given the increase in hatching and survival rates which can be achieved. The design of an incubator and water 31

32 flow adjustments depend on the species, egg density, adhesiveness and susceptibility to mechanical shock. A biological knowledge of the species requirements is essential, however the technical principles are simple which are supply good quality water at a constant temperature, incubate in low light and prevent mechanical damage to the eggs by providing gentle water flow over the eggs (Carral et al., 1992) Factors affecting egg incubation Based on a review article written by Peters (1983), spawning of broodstock, embryo development, survival and growth of fish larvae occur within a narrow range of water temperatures. Incubation temperature has a direct effect on the timing of embryonic development and thus determines hatch rate. Fish development and hatching is delayed at low temperatures and accelerated at high temperatures. Incubating temperatures are also known to modify the behavior of larvae and determine certain morphological characteristics. If a species optimum water temperature for incubation is unknown, use the optimum temperature of a related species or of a fish that inhabits a similar geographic area. In general, optimum temperatures for spawning, incubating and rearing newlyhatched tropical ornamental freshwater species are 24 to 28ºC. The development process from fertilized egg to hatching, like all other biological processes, is dependent upon water temperature; i.e. the higher the water temperature the faster the eggs hatch. The best water temperature for incubation ranges from 25 to 30 C which guarantees the production of strong larvae within a reasonably short time. It is important to protect the eggs from fungal infection during incubation. Every 6 to 12 hours, the eggs should be treated with malachite green, at a concentration of about 6 mg/l. A stock solution is prepared containing 500 mg/l malachite green. The water flow is stopped in the jars to be treated, and 10 ml of the stock solution are poured into each 7-litre 32

33 jar. The chemical is thoroughly mixed in the jar and care being taken not to damage the eggs. After about 5 minutes, the water supply is reopened and the chemical gradually washes out (Little et al., 1987). When the fertilization of the eggs has not been very successful, a layer of white dead eggs gradually accumulates above the fertilized eggs. Soon they will be covered with the fungus Saprolegnia which endangers the fertilized eggs. In such cases, when the eggs have reached the eyed embryonic stage at the end of the second day of incubation, the water flow should be stopped and the eggs allowed to settle down at the bottom of the jar. The ring of white eggs at the top of the other eggs can now be clearly seen. The dead eggs are carefully siphoned out of each jar, and the water flow is reopened. This operation should be repeated about 10 hours later, if necessary, before the hatching of the eggs occurs (Little et al., 1987). The amount of light received during incubation can affect both fish development and larval survival. Incubation of fish embryos should be done either in dim light or darkness. Many species of fish will not hatch in day-light, therefore, if the lights are switched off, hatching will occur a few hours later (Perez et al., 1999). During incubation, a constant water flow is essential for preventing accumulation of waste products and allowing gas exchange between the eggs and the surrounding water. Constant motion also appears to be necessary for successful hatching for some species of fish. Proper water flow also may reduce mechanical abrasion. Eggs of many fish are very sensitive to mechanical shock and should not be moved during certain times while in the development stage (Rana, 1988). Egg diameter also is an important consideration during incubation. Screen mesh size should prevent the passage of eggs while allowing sufficient water circulation and deterring debris collection. Most ornamental fish eggs are around 0.8 mm in diameter. However, the 33

34 size range is wide. For example, eggs can be as large as 1.5 to 2.0 mm for some ornamental tilapia and as small as 0.4 mm for gobies (Rana, 1986). In conclusion, during the incubation period, the water flow should be controlled and regulated. The water temperature should be checked, the development of the eggs should be closely followed and the malachite-green treatment should be administered according to the schedule. It is essential to inspect the working jars regularly day and night because such close and constant supervision will help to avoid important losses in production Larval Rearing The new hatchlings can be kept in the incubators and do not have to be fed as they rely on the food resource within their yolk sac. Healthy larvae tend to stay in dark places and should not be exposed to direct sunlight. After 3 to 4 days the yolk sac will be absorbed and the hatchling is visibly developed into a small tilapia. At this stage the hatchling must be fed on external food for its further development and survival. Therefore, the hatchlings should be transferred out of the incubation facilities to small aquariums or growth tanks. This phase of rearing from first feeding larvae to fingerling size is usually carried out either within growth tanks or in specialized batch aquariums. Food is distributed once a day in the morning or preferably several times daily and the artificial food is scattered over the water surface in the rearing tanks. During the first rearing period, the growth, survival and health of the growing fry should be regularly controlled. The fry may be observed directly in the tank, using a white plate as a background. The survival rate is an important data in order to make any conclusion regarding the induction spawning by using ovaprim on red tilapia (Little and Hulata, 2000). 34

35 Chapter MATERIALS AND METHODS 3.1 INTRODUCTION This study was carried out to determine the spawning performance of mature red tilapia fish with different body weights and body lengths. The females were administered with ovaprim of 1.0 ml/kg body weight via two separate intramuscular injections. Earlier studies in our laboratory, have indicated that 1.0 ml/kg of ovaprim showed to produce the maximum number of eggs (Farizah, 2007). Number of eggs produced, total weight of eggs, weight of one egg, diameter of eggs, hatching rate and fry survival rate were recorded. Blood samples were collected from the fish in order to determine the levels of oestradiol. Comparison between artificial and maternal incubations was made to determine which incubation technique could result in higher hatching and fry survival rates. Experiments were carried out at the Fish Breeding Laboratory, Genetics Building, University of Malaya from October 2006 until April EXPERIMENTAL FISH Red tilapias (Oreochromis niloticus) were used in this study. The broodstocks were obtained from Pusat Perikanan Air Tawar Bukit Tinggi, Bentong Pahang. They were categorized into three different groups of body weight: <300 g (Group I), 300 to 450 g (Group II) and >450 g (Group III). All groups of broodstocks were considered mature and ready to be induced. Besides the above groups, they were also categorized based on body length: 22 to 24 cm (Group A), 25 to 27 cm (Group B) and 28 to 30 cm (Group C). Mature male tilapias were also used for spawning purposes to facilitate fertilization. 35

36 All tilapias were stocked in 2000 litre tanks with 1500 mm diameter, 700 mm inlet height and 1000 mm total height with central drainage. All tanks were supplied with adequate oxygen supply through water-aeration. Water in each of the tank was replaced three times a week to give hygienic and well aerated water to the fish. This may also prevent any stressed conditions and illnesses to the fishes. Common salt has been used as water treatment against any parasitic infections and chlorinated water source. The tilapias were acclimatized between 2 to 7 days before ovulation induction could be carried out (Myers and Hershberger, 1991). The tilapias were fed once daily with high protein fish pellet (Dindings Malaysia) at a rate of 5% of fish biomass. Water maintenance was closely monitored to ensure no appetite disturbance due to water irritation and illness (Legendre, 2000). 3.3 BROODSTOCK SELECTION AND MANAGEMENT Selection of brooding females of red tilapia was based on general shape of the body, scale distribution, state of health and development of sexual organs. In particular, the selected fish should be in good health condition, with no body wounds, no parasites, a typical scale distribution and no fin or body deformation. The tilapias were also checked for their sex, maturity and readiness to produce eggs or semen by visual examination of their genital papilla. Tilapias show a large degree of sexual dimorphism compared to most other fish species in aquaculture. The males and females can be identified on the basis of external genital morphology (Balarin, 1979) at approximately 10 g body weight. The males were selected based on pointed and reddish papilla, while selected females were based on round and reddish papilla, softness of abdomen and uniform size of intra-ovarian oocytes (Beveridge and McAndrew, 2000). Fine transverse vaginal opening and reddish papilla through visual examination is a good indicator for a spawning broodstock. Checking by 36

37 application of gentle pressure on the male abdomen and expressing a small volume of milt can be used in maturity selection (Yang and Kwak, 2004). The technique required the operator to assess the number of opening in the urogenital papillae of each fish. Male and female red tilapias were differentiated by the shape of the body and relative position of the genital papilla. The external differences between the sexes are based on the fact that the male has two orifices under its belly, one is the anus and the other the urogenital aperture. The female has three, which are; the anus, the genital and urinary apertures (Figure 2.2). The anus is easily recognized as a round hole. The urogenital aperture of the male is a small point and the urinary orifice of the female is microscopic and is scarcely visible to the naked eye, while the genital orifice is an opening in a line perpendicular with the axis of the body (Beveridge and McAndrew, 2000). After conditioning, the female breeders were checked for their readiness to spawn by examining their morphological characteristics visually. They were then categorized into one of the following maturity conditions: ready to spawn (RS), swollen (S), not ready to spawn (NRS), and has spawned (HS). Descriptions of these four categories are given in Table 3.1. The category of ready to spawn was first selected for ovaprim induction and pairing with males in a breeding tank. 37

38 Table 3.1: Different categories of sexual maturity of female red tilapias Morphological Days to spawn Category Code characteristics (days) Pink to red and protruding genital Ready to spawn RS papilla, fully opened 3 to 7 genital pore and distended abdomen Pink to yellow genital papilla, Swollen S slightly opened 5 to 10 genital pore and slightly distended abdomen White to clear and Not ready to spawn NRS flat genital papilla 21 to 30 and normal abdomen Red genital papilla Has spawned HS and shrunken to 15 to 30 compressed abdomen 38

39 Female and male red tilapias were kept in separate broodstock tanks in order to prevent wild spawning. Multiple sets of tanks were used for ripening males and females (Figure 3.3). The female tanks were classified according to the three different groups of body weight and body length broodstocks which were Group I, Group II and Group III as well as Group A, Group B and Group C, respectively. This method also was used to simplify calculations of ovaprim dosages for each group of body weight. The breeders were taken out from the storage tanks one day before artificial propagation was carried out. They were crowded into a well aerated tank. The breeders were carefully selected one by one whereby breeders with mature and visible genital papilla were placed into a stocking tank while immature breeders were placed back into the storage tanks. Unhealthy fish were sent to isolated aquariums for treatment. This selection process is important especially for females whose maturity should be checked thoroughly to ensure the success of the semi-artificial propagation. 39

40 Figure 3.1: Selection and stocking brooders for spawning induction. Figure 3.2: Acclimatization of brooders in spawning tank conditions. 40

41 3.4 OVAPRIM DOSAGE A volume of 100 ml per self-sealing bottle of ovaprim was used to induce the spawners. Ovaprim was packaged ready for use in clear liquid form. Precise amounts of individual injection were extracted safely from the bottle directly into a syringe. Each individual female body weight was recorded before intramuscular injection. The weight of the fish will determine the amount of ovaprim required for injection. Amount of injection = Ovaprim dosage (ml/kg) x Fish weight (kg) For example, the recommended dosage of ovaprim is 1.0 ml/kg body weight and the fish weight is approximately 500 g each. Then the desired amount of injection is equal to 1.0 ml/kg multiplied by 500 g, which is converted to 0.5 kg (0.5 ml ovaprim per fish). It was a practice to add 20% to these calculated quantities of ovaprim to compensate for the losses occurring during the injections. Effective dosage of 1.0 ml/kg ovaprim was the standard dosage used in this hormonal induction experiment. Based on dosages of hormonal induction, 1.0 ml/kg ovaprim has been shown to be the effective dosage on stimulating ovulation. This has been shown earlier in experiments in our laboratory that is by using different hormonal treatments which were 0.5 ml/kg, 0.6 ml/kg, 0.7 ml/kg, 0.8 ml/kg, 0.9 ml/kg, 1.0 ml/kg, 1.1 ml/kg, 1.2 ml/kg and 1.3 ml/kg. These dosages were tested for all types of body weight of matured female tilapias. It was shown that 1.0 ml/kg to be the most effective dosage (Farizah, 2007). Therefore, all three groups of tilapias were treated with standard calculation based on 1.0 ml/kg effective dosage ovaprim. Amount of injection = 1.0 ml/kg x 0.5 kg = 0.5 ml 41

42 3.5 SPAWNING INDUCTION It was ensured that all equipment was clean and, if possible, sterilized. In the loading process, ovaprim that was only required for the weight of the fish was withdrawn from the bottle. The needle was pointed upward and the syringe was squeezed gently to expel any trapped air. Then the syringe was pulled slowly to extract the fluid into the syringe until the required amount was obtained. The proper injection procedure is very important to prevent any loss of administered hormone. Intramuscular injections were done in between the dorsal upper part of the fish above the lateral line and below the anterior part of the dorsal fin. During injection, a brood fish was placed in a cloth clamp. The fish was let to lie laterally in a fish holder. Then a syringe needle was pointed towards the head of the fish at an angle of 45º to the body s longitudinal axis, followed by insertion of the needle through the muscle to a depth of about 1.5 cm or to the fish vertebral. The fluid was then injected slowly, and the needle quickly removed. It is very important to place the needle properly so that it slides under the scale rather than through it (Rottmann and Shireman, 1991; Sridhar, 1998 and Hill, 2005). The induced fish was gently placed into a spawning tank of fresh aerated water. Hormone calculations and number of injections are shown in Appendix Table 1.2. Two dosage levels were used as a preparatory dose and a decisive or final dose with a time gap generally of 12 to 24 hours between the two injections (Hill, 2005). In this experiment, the first dosage was injected with 20% of ovaprim and the second injection with 80% of ovaprim. Each respective dosage must be added with an additional 20% of ovaprim to replace any loss of the hormone. The first injection was done on the first day in the evening at 1700 hours and the second injection was done on the subsequent morning at 0800 hours. The interval between these 2 injections should be 15 hours. The first injection was done to promote maturation of the eggs and the second one was to induce spawning 42

43 (Xu, 1999). The injected females were placed back into the spawning tanks supplied with well-clean-aerated water. At the end of the ovulation period, which was about 1 hour from the last injection, mature males were added to each of the spawning tanks. They would then closely interact and prepare their territory for mating with mature females which lead to spawning. Observation was done for every 3 hours interval time, which was at 0800 hours, 1100 hours, 1400 hours and 1700 hours. Table 3.2: Summary of ovaprim induction procedures Day Time Activity st injection of 20% ovaprim hours nd injection of 80% ovaprim hours Blood sampling (for 3 consecutive days) Pairing and observation for spawning hours 2 to 3 Every 3 Observe egg production and timing of egg production hours interval Observe and count eggs produced from each brooder Incubation of eggs under artificial incubator 3 to 6 Every 3 hours interval 6 to 13 Every 3 hours interval Observe any formation of eyed-egg and hatched eggs Calculate hatching rate Observe survival of larva Calculate survival rate 43

44 Figure 3.3: Spawning tanks for ovaprim induction at the hatchery laboratory. Figure 3.4: Intramuscular injection of ovaprim. 44

45 3.6 DETERMINATION OF REPRODUCTIVE CHARACTERISTICS Observation was done everyday during daytime starting from 0800 hours to 1700 hours for checking any ovulatory or spawning response. In other words, observation was done at every 3 hours interval after the second injection during daytime at 0800 hours, 1100 hours, 1400 hours and 1700 hours for checking tilapias ovulatory response (adapted from Yang and Kwak, 2004). Egg production for hormone treatment was determined by counting the amount of eggs produced and recording the total weight of eggs, weight of one egg and diameter of eggs. All data regarding egg number, total weight of eggs, weight of one egg and egg diameter were recorded before artificial or maternal incubation in which hatching rate and fry survival rate were calculated. The eggs produced were collected carefully from the spawner s mouth (except for maternal incubation) by forcing the brooder itself to expel out all the eggs using index finger to open up the mouth. This technique needed gentle handling inside a collector basin which contained water to prevent stress and egg loss. All the eggs from the brooder s mouth were placed into an artificial incubator connected to a continuous water source. Before placing the eggs into the artificial incubator, they were rinsed by using fresh water at least twice to discard them from any waste materials or fungal contamination. The mean diameters of viable eggs were recorded by using a ruler in millimeter scale and were weighed in a plastic bowl which had previously been weighed empty by using Mettler- Toledo weighing scale. Then, the eggs were also calculated by using a tally counter to analyze the hatching and fry survival rates. All significant data were collected at this time. The timing of spawning and all the reproductive parameters (egg number, total weight of eggs, weight of one egg and egg diameter) were recorded and subsequently maintained in an artificial incubator (hatchery facilities) and maternal incubation (natural) until swimming larva were observed (adapted from Yang and Kwak, 2004). 45

46 Figure 3.5: Maternal brooders with incubated eggs. Figure 3.6: Rinsing eggs collected from broodstock s mouth for artificial incubation. 46

47 Figure 3.7: Incubation of viable eggs in an artificial incubator. Figure 3.8: Measuring diameter of fish egg. 47

48 Figure 3.9: Fungal contamination in artificial incubation. 48

49 3.7 ARTIFICIAL INCUBATION AND MATERNAL INCUBATION TECHNIQUES ON DETERMINATION OF HATCHING RATE AND FRY SURVIVAL RATE Egg incubation was done in dechlorinated water (adapted from Yang and Kwak, 2004). The incubation of the eggs was done by modification of a small aquarium, water filter pump and floating square plastic box with sided mesh plate (Figure 3.11). The amount of water flow passing through the mesh plate was maintained during the incubation period for development of the eggs. The artificial incubators were completed with slow circulation of water flow to give aeration and slow movement to the eggs. The eggs were placed inside the incubator. The batches of eggs produced were separated by attaching labels to each incubator. Water temperature for egg incubation in hatchery laboratory ranged from 22 C to 25 C (room temperature) which had been observed by using thermometer. For every 6 to 12 hours, the eggs were treated with malachite green, at a concentration of about 6 mg/l in order to protect the eggs from fungal infection during incubation. A slightly higher concentration of malachite green (6 mg/l) was used due to heavy fungal contamination in the laboratory water reserve at the particular time which can be fatal to the eggs. When the fertilization of the eggs had not been very successful, a layer of white dead eggs gradually accumulated amongst the fertilized eggs. Soon they would be covered with fungus which would endanger the fertilized eggs. Hence, the dead eggs were carefully taken out from each incubator. This operation was repeated at about 10 hours later, for example, before the hatching of the eggs occurs. The amount of dead eggs obtained helped to calculate the hatching rate. Pigmentation rates, the proportion of eggs with pigment cells scattered over the yolk sac (adapted from Galman and Avtalion, 1980), were determined 2 days after spawning as 49

50 an indicator of fertilization rates. Then hatching and survival rate was calculated. The survival rates were calculated based on the amount of survived fry up to 14 days over the amount of hatched fry. The calculation of fertilization rate, hatching rate and survival rate was formulated in order to differentiate developments of eggs during the incubation periods. It was also to measure the performance rates in fertilization success, hatching success and survival success. The calculation of hatching rate was more appropriate than hatchability because the ability to measure the success of hatching process from the formation of pigmented (unhatched) eggs. These 3 levels of reproductive performance in percentage were summarized in the calculation below. Calculation : Fertilization rate : Amount of fertilized eggs (pigmented eggs) x 100 Total amount of eggs Hatchability : Amount of viable eggs x 100 Total amount of eggs Hatching rate : Amount of hatched fry x 100 Amount of fertilized eggs Survival rate : Amount of lived fry up to 14 days x 100 Amount of hatched fry A control experiment was conducted for maternal incubation of eggs. It was done after the spawning event took place. The spawned fishes with full-mouth of brooded eggs were isolated in separate tanks to ensure successful egg incubation. The hatched fry was harvested within 12 days from the first day of spawning. The number of fries produced, were compared with the number of fries produced under artificial incubation for each different body weight groups and body length groups. 50

51 The fry were incubated about 3 to 4 days in artificial incubators until yolk absorption was completed and then transferred to a small aquarium for observation of survival rate. Calculation of the survival rate was done 7 days after stocking the fry in a small aquarium. At the same time, fry from maternal incubation were also being harvested and placed into a glass aquarium with adequate food supply and aeration. Again, hatching rate in artificial incubation and fry survival rate for both incubations were recorded for statistical analysis among the body weight groups, body length groups and between artificial and maternal incubation. 51

52 Figure 3.10: Artificial incubators in the hatchery laboratory. Figure 3.11: Water circulation system in an artificial incubator. 52

53 3.8 BLOOD SAMPLING AND HORMONE ANALYSIS USING RADIOIMMUNOASSAY TECHNIQUE Blood sampling was done to collect serum for investigation of oestradiol fluctuation within the induced spawning period by using the radioimmunoassay protocol. Blood samples were taken at 4 hours interval from fish right after the second injection of ovaprim in the morning (0800 hours), afternoon (1200 hours) and evening (1600 hours) for 3 consecutive days. Blood was removed from the caudal vein which is on the dorsal (upper) part of the fish above the lateral line and below the anterior part of the dorsal fin of each fish (Figure 3.12) by using Terumo Syringe with needle [3 cc/ml with 23 G x 1¼ needles (0.65 mm x 32 mm)] and serum vacutainer tubes (6.0 ml-without heparin). The serum was separated by centrifugation at 3000 rpm for 15 minutes. The separated serum was transferred into individual microcentrifuge tubes (1 ml) and kept at -40ºC before radioimmunoassay was carried out for the oestradiol analysis. Serum oestradiol levels of induced red female tilapias were measured by the RIA protocol at the Animal Biotechnology-Embryo Laboratory (ABEL), Institute of Biological Sciences Principle of Oestradiol Assay Estimation Oestradiol assay estimation was based on the antibody-coated tubes. The 125 I labeled E 2 competes with the respective E 2 for antibody sites in the animal s blood sample. After incubation using a water bath, separation of the bound from the free was achieved by decanting whereby the bound antibody complex remained in the tubes. The tubes were then counted using a gamma counter (1470 Wizard, Wallac, Finland) (Figure 3.13). 53

54 3.8.2 Materials and Reagents for Oestradiol Assay The materials for the oestradiol assays were polypropylene tubes coated with rabbits antibodies to oestradiol in purple coloured, micropipette (100 µl tips for 100 µl), vortex mixer, decanting rack, water bath and gamma counter. Reagents for the oestradiol assay was supplied in the Coat-Count Kit that was purchased from Diagnostic Products Corporation (DPC), USA. One vial of 125 I Oestradiol (105 ml) in liquid form was ready to use. A set of 7 vials with preservatives, labeled A to G respectively containing 0, 20, 50, 150, 500, 1800 and 3600 picograms of synthetic oestradiol per milliliter (pg/ml) in processed human serum. Intermediate calibration points may be obtained by mixing calibrators in suitable proportions. All reagents were stored in a refrigerator at 4ºC and could be used for at least 30 days after reconstitution or before the expiry date RIA Protocol for Oestradiol Quantitative determination of serum oestradiol was carried out using radioimmunoassay kits and was based on a competitive reaction (Imada et al., 2002). All components were stored at room temperature (25 C) before use. A) The oestradiol tubes were labeled as follows: (a) Two plain polypropylene tubes (12x75 mm) were labeled as TC (Total Count) and NSB (Nonspecific Binding) (Tube nos. 1 and 2). (b) Seven oestradiol antibody-coated tubes were labeled as A, B, C, D, E, F and G (Tube nos. 3 to 9) as shown in Table

55 Table 3.3: The arrangement of oestradiol antibody-coated followed by the oestradiol concentration (pg/ml) Calibrators Tube number Oestradiol (pg/ml) A (MB) 3 0 B 4 20 C 5 50 D E F G (c) Three control tubes were labeled respectively as LOW, MEDIUM and HIGH control of serum (Tube nos. 10 to 12). (d) Samples (serum) were labeled as unknown tubes (Tubes nos. 13 onwards). B) Preparation of solutions in NSB, standard, control and unknown tubes (oestradiol) in the pipetting process using pipetting kits: The zero calibrator A (100 μl) were pipetted into the NSB (tube no.2) and A tube (tube no. 3). (a) Each of the remaining calibrators B through G (100 µl) was pipetted into correspondingly labeled tubes (tube nos. 4 to 9). (b) Control (100 µl) and unknown samples were pipetted into their designated tubes (tube nos. 10 to 12 and tube nos. 13 onwards for unknown samples). 55

56 (c) 125 I Oestradiol (1.0 ml) were added to every tube by using the repeater pipette. The tubes were mixed using a vortex mixer for 5 seconds. All solution were pipetted directly to the bottom of the tubes. Disposable micro-pipette tips were used between samples in order to avoid cross contamination. Samples and standards were incubated using a water bath for one hour with 125 I labeled oestradiol as a tracer in antibody-coated tubes. After the incubation process, the contents of the tubes were aspirated and the bound radioactive complexes measured to determine serum oestradiol concentrations with the aid of a Gamma counter. The sensitivity of the assay during oestradiol determinations was 0.03 ng/ml and 3 pg/ml respectively. The intra- and inter-assay coefficient of variation was 7.2% and 7.9% for the oestradiol determination. The supernatants from all the tubes except TC tube were decanted thoroughly using a foam decanting rack and were drained for 2 or 3 minutes. A highly absorbent paper was used to blot the lips of those tubes so that capillary action would remove the last drop of the liquid. The radioactivity in each tube was counted for 1 minute by using the automatic gamma counter. Finally, evaluations of oestradiol concentrations in the unknown serum samples were performed automatically using the MultiCalc Software (Wallac, Finland) via interpolating from the standard curve using a logic-log transformation of the percentage of maximum bound versus the concentration of the standards. 56

57 Figure 3.12: Technique of blood sampling. Figure 3.13: MultiCalc software with computer (Wallac, Finland). 57

58 3.9 EXPERIMENTAL DESIGN This research was divided into 3 experiments: 1) effects of body size (body weight and body length) and ovaprim induction on egg characteristics in relation to oestradiol levels (Experiment 1); 2) effects of artificial incubation on hatching and fry survival rates with reference to body size (body weight and body length) and ovaprim induction (Experiment 2); and 3) effects of maternal incubation on fry survival rates with reference to body size (body weight and body length) and ovaprim induction (Experiment 3) Effects of Body Size (Body Weight and Body Length) and Ovaprim Induction on Egg Characteristics in Relation to Oestradiol Levels (Experiment 1) A total of 225 and 75 selected sexually mature female and male tilapias, respectively, were divided into three groups based on body weights: [Group I (<300 g), Group II (300 to 450 g) and Group III (>450 g)] and body lengths: [Group A (22 to 24 cm), Group B (25 to 27 cm) and Group C (28 to 30 cm)]. Each fish group was placed in 5 different tanks with 3 females and a male in each tank. The spawners were injected with ovaprim of a specified dose (1.0 ml/kg body weight) for 2 consecutive days whereby 20% of the dose was given on the first day and the remaining was on the second day. No control (non-ovaprim induction) was included in this experiment since no response in spawning were observed during the earlier experimental periods. Blood samples were randomly taken (3 times a day, i.e. morning, afternoon and evening with one fish per session) from the fish for 3 consecutive days beginning on the second day of ovaprim injection. Oestradiol levels were determined using the radioimmunoassay (RIA) technique. Reproductive parameters (egg number, total weight of eggs, weight of one egg and egg diameter) were obtained on day 3 after ovaprim injection that is by collecting the eggs from the spawners. The experiments 58

59 were replicated 5 times. The effects of body weights and body lengths on the reproductive parameters and oestradiol levels were determined. Correlations among parameters were made Effects of Artificial Incubation on Hatching and Fry Survival Rates with Reference to Body Size (Body Weight and Body Length) and Ovaprim Induction (Experiment 2) This experiment was an extension of Experiment 1 (Section 3.9.1). The eggs obtained from each body weight and body length group of spawned females were incubated with the artificial incubation technique. Hatching rate was determined by counting the eggs hatched out of the total eggs produced from the artificial incubator. Fry survival rate was determined on day 12 after spawning that is by counting the number of viable fries (100%) and recounting 7 days later after being placed in an aquarium. The fry survival rate was determined from the data obtained for each group of body weight and body length Effects of Maternal Incubation on Fry Survival Rate with Reference to Body Size (Body Weight and Body Length) and Ovaprim Induction (Experiment 3) This experiment was an extension of Experiment 1 (Section 3.9.1). The eggs obtained from each body weight and body length group of spawned females were incubated into maternal incubation. The fry survival rate was determined on day 12 after spawning that is by counting the number of viable fries (100%) and recounting 7 days later after being placed in an aquarium. The fry survival rate was determined from the data obtained for each group of body weight and body length. Hatching rate was not determined due to technical problems in obtaining the data. 59

60 Mature red female tilapias EXPERIMENT 1 Body weight (Groups I, II and III) and Body length (Groups A, B and C) Ovaprim induction Blood sampling RIA protocol E 2 analysis Reproductive parameters Number of eggs Total weight of eggs Weight of one egg Diameter of eggs EXPERIMENT 2 Artificial incubation Hatching rate Fry survival rate EXPERIMENT 3 Maternal incubation Fry survival rate Figure 3.14: Flowchart of experimental design. 60

61 3.10 STATISTICAL ANALYSIS All data were assessed by Analysis of Variance (ANOVA) and Multiple Range Test (DMRT) using the software package SPSS (Statistical Package for Social Science). All parameters measured such as egg characteristics (number of eggs, total weight of eggs, weight of one egg, diameter of eggs), hatching rates and fry survival rates were compared and analyzed in terms of breeding performance among the group weights, lengths and type of incubation as well as the oestradiol levels among different sizes, days and time of the day. For all statistical tests, values were expressed as mean±sem and considered significantly different with P values of less than 0.05 (P<0.05). 61

62 Chapter RESULTS 4.1 EFFECTS OF BODY SIZE (BODY WEIGHT AND BODY LENGTH) AND OVAPRIM INDUCTION ON EGG CHARACTERICTICS IN RELATION TO OESTRADIOL LEVELS (EXPERIMENT 1) For this experiment, 5 spawning tanks were used for each group of body weight or body length with 3 matured induced female fish per tank. 5 trials were repeated for each group of body weight or body length. Table 4.1 shows that number of eggs per female and total weight of eggs were significantly higher in both large and medium body weights of tilapia fish compared with the small body weight. The corresponding average values were ±158.5, ±187.9 and 276.2±39.5 eggs per female and 10.39±1.23, 11.88±1.99 and 3.32±0.52 grams, respectively. There were no significant differences in weight of one egg and diameter of eggs among the large, medium and small body weights (range: 0.009±0.001 g, 0.010±0.001 g and 0.011±0.001 g; 2.25±0.08 mm, 2.40±0.07 mm and 2.17±0.17 mm, respectively). As for short, medium and long body lengths, there were significant differences in weight of one egg of the induced red female tilapia fish. They were found to be higher in the shorter and medium length fish. The average values are as follows: 0.012±0.001, 0.011±0.001 and 0.008±0.001 grams, respectively. Meanwhile there were no significant differences in number of eggs per female, total weight of eggs and mean diameter of eggs among the short, medium and long body lengths (range: 687.3±144.9, ±215.7 and ±214.8 eggs per female; 6.70±1.23, 12.13±2.13 and 8.11±1.50 grams; 2.33±0.17 mm, 2.20±0.08 mm and 2.35±0.08 mm, respectively). 62

63 The interactions between body weights and body lengths of the fish exhibited no significant differences in diameter of eggs. However, these interactions showed significant differences on number of eggs, total weight of eggs and weight of one egg, whereby interactions of large body weight with long body length (1398.0±235.8 eggs per female) and large body weight with medium body length (1388.7±385.5 eggs per female) produced higher number of eggs among the body weight and body length groups. Medium body weight with medium body length (14.80±2.85 g) and large body weight with medium body length (13.33±3.18 g) produced higher total weight of eggs among the body weight and body length groups. Small body weight with small body length (0.014±0.001 g) showed higher weight of one egg among the body weight and body length groups. Meanwhile, the interactions for all small body weight groups with short, medium and long body lengths exhibited the lowest number of eggs and total weight of eggs. The respective values are as follows: 267.5±124.5, 283.0±69.0 and 278.0±55.0 eggs per female; 3.50±1.50, 3.65±1.15 and 2.80±0.20 grams. As the interactions for small, medium and large body weight groups with long body length group exhibited the lowest weight for one egg. The respective values are as follows: 0.009±0.001, 0.008±0.001 and 0.007±0.001 grams. 63

64 Table 4.1: Number of eggs per female, total weight of eggs, weight of one egg and diameter of eggs (mean±sem) for interaction between weights and lengths of tilapia fish induced by ovaprim No. of eggs per female Total weight of eggs (g) Weight of one egg (g) Weight of fish * (g) Weight of fish * (g) Weight of fish * (g) Diameter of eggs (mm) Weight of fish * (g) Length ** of fish (cm) Mean Mean Mean Mean A ± a,x ± b,x ± 21.5 b,x ± x 3.50 ± 1.50 a,x 9.50 ± 1.50 b,x 7.10 ± 0.20 ab,x 6.70 ± 1.23 x ± a,y ± a,y ± a,y ± y 2.50 ± 0.50 a,x 2.50 ± 0.00 a,x 2.00 ± 0.00 a,x 2.33 ± 0.17 x B ± 69.0 a,x ± a,x ± a,x ± x 3.65 ± 1.15 a,x ± 2.85 a,x 13.3 ± 3.18 a,x ± 2.13 x ± a,x ± a,y ± a,xy ± y 2.00 ± 0.00 a,x 2.30 ± 0.12 a,x 2.17 ± 0.17 a,x 2.20 ± 0.08 x C ± 55.0 a,x ± a,x ± a,x ± x 2.80 ± 0.20 a,x 8.60 ± 4.16 a,x 9.94 ± 1.13 a,x 8.11 ± 1.50 x ± a,x ± a,x ± a,x ± x 2.00 ± 0.00 a,x 2.50 ± 0.00 b,x 2.40 ± 0.10 b,x 2.35 ± 0.08 x Mean ± 39.5 a ± b ± b 3.32 ± 0.52 a ± 1.99 b ± 1.23 b ± a ± a ± a 2.17 ± 0.17 a 2.40 ± 0.07 a 2.25 ± 0.08 a a, b Means within a row within a group with different superscripts were significantly different at (P<0.05) x, y Means within a column within a group with different superscripts were significantly different at (P<0.05) * Weight of fish : 1 = <300 grams, 2 = 300 to 450 grams and 3 = >450 grams ** Length of fish : A = 22 to 24 cm, B = 25 to 27 cm and C = 28 to 30 cm 64

65 Through statistical correlation analysis (Appendix Table 4.16), it was shown that the correlation was positively significant between fish body weight and number of eggs produced (P<0.01) as well as between fish body weight and total weight of eggs (P<0.05). Meanwhile fish body weight and weight of one egg showed negatively significant correlation between them. Larger body weight tended to produce high number of eggs and subsequently would increase the total weight of the eggs in which indicated that the body weight could have effect on the production of eggs in a single spawning. Unfortunately, larger body weight showed lowest mean weight of one egg indicating that the larger fish were able to produce higher number of eggs but reduced in mean weight of one egg. There was negative correlation between body lengths with weight of one egg (P<0.01) and the number of eggs showed significant positive correlation with total weight of eggs (P<0.01). When the coefficient correlation was carried out, there was a highly significant correlation (r = 0.846) (P<0.01) between body weight and body length. The mean concentrations of oestradiol (E 2 ) were ± pg/ml in small body weight, ± pg/ml in medium body weight and ± pg/ml in large body weight of tilapia fish. There was a significant difference in E 2 levels between small body weight and medium body weight tilapia (P<0.05) if compared with large body weight tilapia fish (Table 4.2). There were no significant differences in E 2 levels for days after ovaprim injections (day 1, day 2 and day 3) and time of day (morning, afternoon and evening). The respective values are as follows: ±26.382, ± and ± pg/ml; ±12.761, ± and ± pg/ml (Table 4.3). The concentration of E 2 did not change from day 1 to day 3. Day 2 produced the highest concentration of E 2 during the events of ovulation, from 77.54± pg/ml on day 1 to 81.42± pg/ml on day 2. On day 3, the E 2 concentration decreased to 65

66 68.06±76.14 pg/ml. Analysis of variance showed no significant effect of day on E 2 concentration (Appendix Table 3.4). E 2 concentrations in the morning (54.50±76.56 pg/ml), afternoon (94.05± pg/ml) and evening (78.47±99.67 pg/ml) were not statistically significant. There were no significant differences in the interaction between body weight groups and days after ovaprim injections (Table 4.2) for concentration of E 2. However, medium body weight tilapia on day 1 showed the highest concentration of E 2 ( ± pg/ml); and the lowest E 2 concentration was the small fish body weight in day 1 (28.16±23.60 pg/ml). In general, medium fish body weight recorded the highest production of E 2 from day 1 to day 3 (341 pg/ml), followed by large fish body weight (199 pg/ml) and by small fish body weight (141 pg/ml). Table 4.2 shows no significant differences between body weight groups and time of day after ovaprim injections. However, medium body weight in the afternoon exhibits the highest concentration of E 2 ( ± pg/ml) as well as the highest total E 2 production in the morning, afternoon and evening (268 pg/ml). Large fish body weight produced 200 pg/ml E 2 in all times of the day and the lowest for small fish body weight in which produced 141 pg/ml E 2 in all times of the day. Table 4.3 shows no significant interactions between days and time of day on E 2 concentrations (range: ± pg/ml to ± pg/ml). Through statistical correlation analysis, concentrations of E 2 in fish body weight showed positive correlations with total weight of eggs (P<0.01) and diameter of eggs (P<0.05). However, concentration of E 2 in fish body length showed no correlations in number of eggs and diameter of eggs, except there was a positive correlation with total weight of eggs (P<0.05). 66

67 Table 4.2: Concentrations of oestradiol (mean±sem) for interaction between weight of fish with day and time of day after ovaprim injections in induced red tilapia fish Oestradiol pg/ml Weight of fish* Day** ±6.811 a,x ± b,x ± ab,x ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x Time of day*** ±7.417 a,x ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x Mean ± a ± b ± ab a, b Means within a row within a group with different superscripts were significantly different at (P<0.05) x Means within a column within a group with same superscript were not significantly different at (P>0.05) *Weight of fish : 1 = <300 grams, 2 = 300 to 450 grams and 3 = >450 grams **Day : 1 = first day of second ovaprim injection 2 = second day after second ovaprim injection 3 = third day after second ovaprim injection ***Time of day : 1 = morning, 2 = afternoon and 3 = evening 67

68 Table 4.3: Concentrations of oestradiol (mean±sem) for interaction between day and time of the day after ovaprim injections in induced red tilapia fish Oestradiol pg/ml Day* Time of day** Mean ± ± ± ± a,x a,x a,x x ± ± ± ± a,x a,x a,x x ± ± ± ± a,x a,x a,x x Mean ± ± ± a a a a Means within a row within a group with same superscript were not significantly different at (P>0.05) x Means within a column within a group with same superscript were not significantly different at (P>0.05) *Day : 1 = first day of second ovaprim injection 2 = second day after second ovaprim injection 3 = third day after second ovaprim injection **Time of day : 1 = morning, 2 = afternoon and 3 = evening 68

69 4.2 EFFECTS OF ARTIFICIAL INCUBATION ON HATCHING AND FRY SURVIVAL RATES WITH REFERENCE TO BODY SIZE (BODY WEIGHT AND BODY LENGTH) AND OVAPRIM INDUCTION (EXPERIMENT 2) With reference to Table 4.4, there were no significant differences on hatching and fry survival rates for small, medium and large fish body weights in artificial incubation. The corresponding average values were 89.50±2.03%, 78.80±3.31% and 78.34±8.13%; and 97.17±0.75%, 93.60±1.41% and 94.80±0.84%, respectively. There were no significant differences on hatching and fry survival rates for short, medium and long fish body lengths in artificial incubation. The corresponding average values were 83.83±4.03%, 85.00±1.69% and 75.54±8.41%, and 93.50±2.06%, 96.50±0.60% and 94.10±1.07%, respectively. From Table 4.4, the interactions between body weights and body lengths of the fish in artificial incubation exhibited no significant differences in hatching rate. However, this interaction showed significant differences in fry survival rate, whereby medium body weight and short body length exhibited the lowest fry survival rate (88.00±2.00%). Meanwhile, small body weight group and its interactions with short, medium and long body lengths exhibited the highest fry survival rate compared with the other body lengths groups interactions. The respective values are as follows: 98.50±0.50%, 97.00±2.00% and 96.00±1.00%. Based on correlation analysis, number of eggs showed negative correlation with hatching rate. Besides that, the hatching rate were positively correlated with the fry survival and weight of one egg, meanwhile it was negatively correlated with E 2 concentration. 69

70 Table 4.4: Hatching rate and fry survival rate (mean±sem) for interaction between weights and lengths of tilapia fish induced by ovaprim in artificial incubation Hatching rate (%) Fry survival rate (%) Length Y of fish (cm) Weight of fish X (g) Weight of fish X (g) Mean Mean A ± ± ± ± ± ± ± ± 5.00 a,x 1.50 a,x 9.00 a,x 4.03 x 0.50 b,x 2.00 a,x 2.00 ab,x 2.06 x B ± ± ± ± ± ± ± ± 6.00 a,x 1.83 a,x 1.45 a,x 1.69 x 2.00 a,x 0.97 a,y 0.88 a,x 0.60 x C ± ± ± ± ± ± ± ± 0.50 a,x a,x a,x 8.41 x 1.00 a,x 3.00 a,xy 1.34 a,x 1.07 x Mean ± ± ± ± ± ± 2.03 a 3.31 a 8.13 a 0.75 a 1.41 a 0.84 a a, b Means within a row within a group with different superscripts were significantly different at (P<0.05) x, y Means within a column within a group with different superscripts were significantly different at (P<0.05) X Weight of fish : 1 = <300 grams, 2 = 300 to 450 grams and 3 = >450 grams Y Length of fish : A = 22 to 24 cm, B = 25 to 27 cm and C = 28 to 30 cm 70

71 4.3 EFFECTS OF MATERNAL INCUBATION ON FRY SURVIVAL RATE WITH REFERENCE TO BODY SIZE (BODY WEIGHT AND BODY LENGTH) AND OVAPRIM INDUCTION (EXPERIMENT 3) As for maternal incubation, there were no significant differences in fry survival rate for small, medium and large fish body weights. The corresponding average values were 94.00±3.21%, 96.50±0.67% and 94.71±2.17%. There were no significant differences in fry survival rate for short, medium and long fish body lengths. The corresponding average values were 91.50±3.30%, 97.33±0.56% and 96.86±0.91% (Table 4.5). The interactions between body weight and body length groups of the fish in maternal incubation exhibited no significant differences in fry survival rate, however, medium and long fish body length groups for the medium and large fish body weight showed significant differences in fry survival rates as compared to others. The values for fry survival rate ranged from 87.00±0.00% to 98.50±0.50%. Based on correlation analysis (Appendix Table 4.16), number of eggs showed negative correlation with hatching rate. Besides that, the hatching rate was positively correlated with the fry survival and negatively correlated with E 2 concentration. 71

72 Table 4.5: Fry survival rate (mean±sem) for interaction between weights and lengths of tilapia fish induced by ovaprim in maternal incubation Weight of fish* (g) Length of fish** (cm) Mean A 92.25±6.76 a,x 94.50±0.50 a,x 87.00±0.00 a,x 91.50±3.30 x B 96.00±1.00 a,x 97.50±0.50 a,y 98.50±0.50 a,y 97.33±0.56 x C 95.50±0.50 a,x 97.50±0.50 a,y 97.33±2.19 a,y 96.86±0.91 x Mean 94.00±3.21 a 96.50±0.67 a 94.71±2.17 a a Means within a row within a group with same superscript were not significantly different at (P>0.05) x, y Means within a column within a group with different superscripts were significantly different at (P<0.05) *Weight of fish : 1 = <300 grams, 2 = 300 to 450 grams and 3 = >450 grams **Length of fish : A = 22 to 24 cm, B = 25 to 27 cm and C = 28 to 30 cm 72

73 4.4 EFFECTS OF BODY SIZE (BODY WEIGHT AND BODY LENGTH) ON THE FRY SURVIVAL RATE FOR COMBINATION OF BOTH ARTIFICIAL AND MATERNAL INCUBATIONS (EXPERIMENTS 2 AND 3) Table 4.6 shows the comparison of fry survival rate for three different fish body weights in artificial and maternal incubations. There were no significant differences in all fry survival rates for small, medium and large fish body weights. The respective values are as follows: 95.36±1.86%, 94.69±0.97% and 94.76±0.98%. The comparison of fry survival rate for three different fish body lengths in artificial and maternal incubations were also shown. There were significant differences in fry survival rates for short, medium and long fish in which the mean of the medium body length were significantly the highest. There were significant differences in fry survival rates in the interactions between body weight and body length groups for both types of incubations. Short fish body length with medium fish body weight and large fish body weight showed the lowest fry survival rates (91.25±2.06% and 90.50±2.18%, respectively). The values of fry survival rate ranged from 90.50±2.18% to 97.40±0.68%. 73

74 Table 4.6: Fry survival rate (mean±sem) for interaction between weights and lengths of tilapia fish induced by ovaprim for combination of both artificial and maternal incubations Weight of fish* (g) Length of fish** (cm) Mean A 94.33±4.48 a,x 91.25±2.06 a,x 90.50±2.18 a,x 92.36±2.03 x B 96.50±0.96 a,x 96.57±0.72 a,y 97.40±0.68 a,y 96.81±0.43 y C 95.75±0.48 a,x 94.80±1.99 a,xy 95.25±1.24 a,y 95.24±0.79 xy Mean 95.36±1.86 a 94.69±0.97 a 94.76±0.98 a a Means within a row within a group with same superscript were not significantly different at (P>0.05) x, y Means within a column within a group with different superscripts were significantly different at (P<0.05) *Weight of fish : 1 = <300 grams, 2 = 300 to 450 grams and 3 = >450 grams **Length of fish : A = 22 to 24 cm, B = 25 to 27 cm and C = 28 to 30 cm In terms of interaction between fish body weights and different type of incubations, Table 4.7 shows no significant differences in all fry survival rates. Fry survival rates in artificial incubation for small, medium and large body weights exhibited more approximately the same effect on fry survival rates in maternal incubation for small, medium and large body weights. The corresponding average values were 97.17±0.75%, 93.60±1.41% and 94.80±0.84%; and 94.00±3.21%, 96.50±0.67% and 94.71±2.17%, respectively. 74

75 In terms of the interactions between fish body lengths and different type of incubations, Table 4.8 shows no significant differences in all fry survival rates. Fry survival rates in artificial incubation for short, medium and long body lengths exhibited approximately the same effect on fry survival rates in maternal incubation for short, medium and long body length. The corresponding average values were 93.50±2.06%, 96.50±0.60% and 94.10±1.07%; 91.50±3.30%, 97.33±0.56% and 96.86±0.91%, respectively. Table 4.7: Fry survival rate (mean±sem) for interaction between weights and type of egg incubation of tilapia fish induced by ovaprim for combination of both artificial and maternal incubations Weight of fish* (g) Type of egg incubation Artificial 97.17±0.75 a 93.60±1.41 a 94.80±0.84 a Maternal 94.00±3.21 a 96.50±0.67 a 94.71±2.17 a a Means within a row within a group with same superscript were not significantly different at (P>0.05) *Weight of fish : 1 = <300 grams, 2 = 300 to 450 grams and 3 = >450 grams 75

76 Table 4.8: Fry survival rate (mean±sem) for interaction between lengths and type of egg incubation of tilapia fish induced by ovaprim for combination of both artificial and maternal incubations Length of fish* (cm) Type of egg incubation A B C Artificial 93.50±2.06 a 96.50±0.60 a 94.10±1.07 a Maternal 91.50±3.30 a 97.33±0.56 a 96.86±0.91 a a Means within a row within a group with same superscript were not significantly different at (P>0.05) *Length of fish : A = 22 to 24 cm, B = 25 to 27 cm and C = 28 to 30 cm As for type of incubation, Table 4.9 shows artificial and maternal incubation exhibited no significant difference in fry survival rates. The corresponding average values were 94.89±0.69% and 94.95±1.39%, respectively. Table 4.9: Fry survival rate (mean±sem) for combination of both artificial and maternal incubations in tilapia fish induced by ovaprim Type of egg incubation Fry survival rate (%) Artificial Maternal 94.89±0.69 a 94.95±1.39 a a Means within a column within a group with same superscript were not significantly different at (P>0.05) 76

77 Fertilized Eggs with Eyed Eggs Stage Dead Eggs with Discoloration Figure 4.1: Formation of eyed egg stage after 3 days incubation. Figure 4.2: Magnification of eyed eggs under microscope. 77

78 Figure 4.3: Hatching larvae after 4 days incubation. Figure 4.4: Body size of newly developing larva. 78

79 Developing Mouth Heart Yolk Sac Figure 4.5: Gross anatomy of developing larva. Figure 4.6: 2 days old fry after hatching in an artificial incubator. 79

80 Figure 4.7: 4 days old fry with formation of mouth. Figure 4.8: 6 days old fry swimming in an artificial incubator. 80

81 Figure 4.9: 7 days old fry in an aquarium for survival observation. Figure 4.10: Surviving and healthy fry within 7 days observation. 81

82 Figure 4.11: 2 weeks old juvenile fish in a rearing tank. 82

83 Chapter DISCUSSION 5.1 EFFECTS OF BODY SIZE (BODY WEIGHT AND BODY LENGTH) AND OVAPRIM INDUCTION ON EGG CHARACTERICTICS IN RELATION TO OESTRADIOL LEVELS (EXPERIMENT 1) In this study, the body weight of the tilapia fish influenced the number of eggs and total weight of eggs after 1.0 ml/kg ovaprim induction. The medium and larger body weights produced higher number of eggs and total weight of eggs compared with the small body weight. However, body weight of tilapia fish did not show any significant effect on weight of one egg and diameter of eggs. As for body lengths, it did not give any major effects in number of eggs per female, total weight of eggs and diameter of eggs of tilapia fish because the differences were not too obvious. Coward and Bromage (1999b) and Masser (1999) reported that there was a strong relationship between fish body weight and fecundity but did not find any significant relationship between egg size and maternal body weight and body length. As for short, medium and long body lengths of the tilapia fish, there were significant differences in weight of one egg. In terms of weight of one egg, there was an inverse relationship between fecundity and total egg weight. The difference in fecundities (seed/female/spawn) could be related to differences in brood fish size and age used (Lim, 2006). Comparisons of tilapia reproductive performance could be a complicated issue because it is affected by brood size, previous spawning history, the production setting and the limitation of broodstock selection. Unfortunately, broodstocks used in the present study were selected randomly by Jabatan Perikanan Bukit Tinggi, Pahang and largely of unknown age. Cruz and Mair (1989) reported that relative fecundity in Nile tilapia has been shown to decrease with increased female size and age. 83

84 However, age by itself may have not been a factor. It stated that medium and large body weights showed higher egg number rather than the large body weight tilapia that should be produced the highest number of eggs than medium body weight tilapia. It is well documented that total fecundity is more related to tilapia size rather than age (Coward and Bromage, 1999a). Guerrero (1994) mentioned that Oreochromis species in natural spawning is a mouth-brooder with small gonads of less than 700 eggs. Macintosh and Little (1995) reported that fecundities ranged from 243 to 847 eggs in all sizes of tilapia. In Tilapia zillii, fecundity increased with increasing fish size (body weight and body length). Furthermore, the present study confirmed that fecundity was significantly related to fish body weight (1314.3±158.5 eggs per female in large body weight tilapia and 276.2±39.5 eggs per female in small body weight tilapia) after 1.0 ml/kg ovaprim induction, a relationship commonly found in teleosts (Coward and Bromage, 1999b). This was confirmed when the analysis showed that correlations were positively significant between number of eggs produced and total weight of eggs (P<0.01). The larger the body weight, the higher the number of eggs produced as well as heavier total weight of the eggs. A wide variation in fecundity was observed among individual fish of similar body weight group. This has been attributed to the effects of age, egg size and genetic factors (Coward and Bromage, 1999b). Overall, there was a trend across body weight for larger weight of females in ovaprim induction to produce higher number of eggs (p<0.01), followed by medium body weight and finally by small body weight of tilapia fish. In relation to the non-significant effect of diameter of eggs (average mean ranged from 2.17±0.17 mm to 2.40±0.07 mm in all body weight groups), this result is consistent with data reported by Singh (1988) where they found no significant differences in egg diameter, percentage hatched and percentage of fry survival among Egypt, Ghana and 84

85 Ivory Coast strains of tilapia fish. This suggests that the different broodstock body weight of the same strain could stock energy and nutrients in eggs normally. Moreover, the results were consistent throughout the whole batches in all trials. The normal development of eggs and hatchlings in the different group of broodstock body weight was also verified by other relative indices, such as fertilization rate, hatching rate and larval normality. Some researchers conclude that egg size variation within and between batches might be possible reasons for phenotypic variation of important traits. Egg size differences might have resulted from epigenetic influences and early acting environment factors such as maternal and ovary developmental differences during vitellogenesis and the ripening of the oocytes (Brummet, 1995). Eventually, similar egg sizes for Nile tilapia were reported by Gunasekera et al. (1996). In addition, Bromage et al. (1991) found that egg diameter to be only poorly related to female body weight. Similarly, Kaeriyama et al. (1995) suggested that egg size was stable within a cohort or population of sockeye salmon (Oncorhynchus nerka) irrespective of variations of body weight. In the present study, fish body weight has shown a significant effect on total weight of eggs in the interaction with fish body length, whereby interaction between small body weight and short body length produced the lowest total weight of eggs. Meanwhile, large body weight and long body length tilapia fish recorded the highest production of eggs but eventually large body weight and long body length recorded the lightest egg mean (0.007 g/egg) compared with small body weight and short body length (0.014 g/egg). Most studies of reproduction tend to consider fecundity and egg weight as separate indicators of reproductive performance. There is an inverse relationship between fecundity and egg weight in the event where fish produce either more eggs of a smaller weight or fewer eggs of a larger weight. It was suggested that total egg volume was a more appropriate index of egg production (Bromage et al.,1992). The reduction of egg weight in fish may be due to 85

86 the lower E 2 levels, which in turn reduces vitellogenin production. The contribution of vitellogenin sequestration to oocyte growth is well organized (Tyler and Sumpter, 1996) as the role of E 2 in stimulating vitellogenesis (Sullivan, 1994). This was reported by Taranger et al. (1999) in a variety of non-salmonid species where plasma levels of E 2 have been recorded in relation to ovarian growth and development and is consistent with the recognized role of E 2 in stimulating hepatic synthesis of the yolk protein precursor vitellogenin. By induction of 1.0 ml/kg body weight ovaprim, larger fish body weight would produce higher concentrations of E 2 in the blood. This would stimulate the development of the eggs prior to ovulation. Therefore, larger fish body weight would produce higher number of eggs and subsequently would increase the total weight of the eggs. However, this vitellogenesis may affect the mean weight of one egg even though it was recorded the higher mean weight of total eggs. Overall, the results from this study do not fully resolve the ambiguity concerning the inverse relationship between fish size and weight of one egg under ovaprim induction because the response in this study would appear to be more of a negative correlation. In addition, there were no effects on diameter of eggs for all body weights in tilapia fish. In this study, by using body weight and body length as separate factors, it showed that there were variations in certain reproductive parameters in spawned tilapia fish even though the coefficient correlation was a highly significant correlation (r = 0.846) (P<0.01) between body weight and body length in all tilapia fish. This might be due to the variations in brooders with respect to their body weight and body length ratios. Some of the spawned tilapia fish showed higher body weight but shorter body length, while in contrast showed smaller body weight but longer body length. This condition have been observed by Negassa and Getahun (2003) which mentioned that poor body weight and body length relationships coincided with time of peak breeding activity in Tilapia zillii. 86

87 In other field of study, there was a research by Ekop et al. (2007) confirmed that mercury content of tilapia decreases with body weight of the fish meanwhile increases with body length. Correlation between the concentration of mercury and the body weight of the fish was negative (r = ) but positive for body length (r = 0.113). Based on this observation, reproductive performance especially in tilapia fish might vary with body weight and body length. Therefore, the consumption of matured and different broodstocks body weight and body length as well as its interactions with the reproductive parameters were advocated in this work. E 2 has been reported to stimulate vitellogenesis in teleosts (Smith and Haley, 1988). Higher levels of E 2 in fish were related to improve reproductive performance compared to fish that had low or reduced levels in which this was associated with a lack of final oocyte maturation (Coward and Bromage, 1998). In the present study, the analysis of variance for E 2 concentrations in tilapia fish after 1.0 ml/kg ovaprim induction (Appendix Table 3.4) exhibited a significant effect (P<0.05) on fish body weight. Medium fish body weight produced the highest E 2 concentration ( ± pg/ml) compared with large (66.409± pg/ml) and small (47.164± pg/ml) fish body weights. Medium fish body weight has a more pronounced effect in E 2 production after 1.0 ml/kg ovaprim if compared with the small fish body weight broodstock. This was compatible with medium fish body weight broodstock which showed higher mean of egg productions and total weight of eggs if compared with the small fish body weight broodstock which showed lower mean of egg productions and total weight of eggs. However, days and time of day showed no significant differences on E 2 concentrations (P>0.05). It can be concluded that fish body weight would influence E 2 production in 1.0 ml/kg ovaprim induction and subsequently would affect fish fecundity. 87

88 The factors of day and time of the day did not influence the production of E 2 in ovaprim induction as well as the interactions between body weight with day, body weight with time of the day and day with time of the day. The mean showed that day 2 (81.424± pg/ml) and afternoon (94.005± pg/ml) showed the highest E 2 production in this trial. The highest level of E 2 on day 2 was compatible with the spawning events in which totally occurred on day 2 in day-light after final ovaprim induction. However, in the statistical analysis, E 2 level was still constant (P>0.05) throughout the day and time of the day along the ovaprim induction in all body weights of tilapia fish. It means that E 2 production by tilapia after ovaprim induction was maintained from day 1 to day 3, even though all of the spawning events occurred on day 2. Based on correlations between the reproductive parameters in Experiment 1, E 2 concentrations does affect the total weight of the eggs produced by the induced broodstocks after 1.0 ml/kg ovaprim stimulation. There was high positive correlation (P<0.01) between E 2 concentrations in the blood with the total weight of the eggs produced after 1.0 ml/kg ovaprim stimulations. This is because higher concentrations of E 2 would increase the egg production and subsequently would increase the total weight of the eggs. Besides that, E 2 concentrations also showed a positive correlation (P<0.05) with diameter of eggs which were produced by the induced broodstocks after 1.0 ml/kg ovaprim stimulations even though diameter of eggs did not give any significant differences in all body sizes of induced tilapia fish in this study. These results concluded that higher concentration of E 2 produced in larger fish body weight would affect the egg production and subsequently would increase the total weight of the eggs as well as the egg size. Likewise, increase in E 2 concentration would eventually decrease fry survival rate. It maybe explained that the artificial incubation system may reduce the percentage of hatching in high number of eggs 88

89 incubated due to competition for the survival requirements of the eggs such as oxygen and space thus reflected to the weakness of fry development and survival. Particular areas of concern include the effect of ovaprim that might influence the reproductive endocrinology of tilapia in which encounter with E 2 action; and thus affect the dynamics of oocyte development and ovulation. This might simply imply that if the interspawning interval (ISI) was longer, eggs had more time to sequester vitellogenin from the bloodstream, hence resulting in a larger final egg size and weight (Specker and Sullivan, 1994). In contrast, the induced tilapia fish with 1.0 ml/kg ovaprim has the shortest ISI and will produce smaller and lighter eggs. This maybe evidence that ovaprim directly affects egg size in this species due to immediate effect in ovulation induction. Shorter time to sequester vitellogenin from the blood stream, resulting in same final egg size among the three body weights of fish even though red tilapia females of larger body weight was found to produce more and bigger eggs (Rana, 1986). In the observations of the present study, 1.0 ml/kg ovaprim was more effective in stimulating egg production rather than affecting the size of the eggs for all body weights of tilapia fish. This might be shorter ISI and resulting in the same final egg size among the 3 body weights of induced tilapia fish. These results showed that the pattern of serum E 2 in these fish were not similar to each other even within the same body weight group. The profile of plasma E 2 concentration in the fish during oocyte maturation and ovulation is still unclear. However, change in plasma E 2 levels was generally correlated with oocyte development in the ovary and increases in gonado-somatic index (GSI). A peak in plasma E 2 levels on day 2, as the spawning event takes place, within the afternoon for induced fish, suggests that the tilapia ovaries and other tissues were receptive to 1.0 ml/kg ovaprim stimulation. The level of plasma E 2 begins to increase in accordance with the appearance of active vitellogenic 89

90 oocytes, and reaches the highest levels in the tertiary yolk stage oocyte in the ovary and sharply declines in fish with postvitellogenic and atretic ovaries (Lee et al., 1998). Through the observations, all 1.0 ml/kg ovaprim induced tilapia fish showed a highly asynchronous nature of E 2 levels even within the same body weight. Possible explanations for this include a shortage of ripe females or unknown spawning background history for induction program. The selected fish might be in the resting stage (not ready to spawn) or just spawned where it would give a negative response to ovaprim stimulation. Coward and Bromage (1998) have stated that the highly asynchronous nature of ovarian recrudescence is part of the flexible reproductive strategy found in Oreochromis species, which can be exploited by certain conditions. For example, the lower levels of E 2 in induced tilapia fish might reflect the higher levels of stress in females maintained constantly in spawning tanks (laboratory conditions). Stress has been previously related to poor reproductive performance and low levels of plasma sex steroids in tilapia. Another possibility to be considered is that the mid-cycle decline in E 2 levels could be due to a rapid utilization of the hormone in stimulating vitellogenesis (Coward and Bromage, 1998). There was a difference in terms of fecundity between fish treated with 1.0 ml/kg body weight ovaprim and control fish. Without ovaprim induction, the fish did not ovulate or asynchronously spawned which showed very poor spawning and fecundity performances. Through 1.0 ml/kg ovaprim induction in this study, synchronization of spawning and higher fecundity was obtained in the laboratory conditions. This also suggests that treatment with ovaprim did not adversely affect the viability of the eggs produced. 90

91 5.2 EFFECTS OF ARTIFICIAL INCUBATION ON HATCHING AND FRY SURVIVAL RATES WITH REFERENCE TO BODY SIZE (BODY WEIGHT AND BODY LENGTH) AND OVAPRIM INDUCTION (EXPERIMENT 2) The present study confirmed that body weight and body length did not affect the hatching and fry survival rates in artificial incubation (P>0.05). Shireman (1991) reported that only a qualitative estimate of egg viability was obtained by observing the hatching of eggs in the incubator. His report has confirmed that there were no significant effects on hatching rate and fry survival rate on body weight and body length in tilapia fish. Small fish body weight that produced the lowest number of eggs incubated artificially, exhibited the highest hatchability if compared with large body weight that produced the highest number of eggs incubated artificially in which exhibited the lowest hatchability. Artificial incubation showed that there was negative correlation between number of eggs incubated with the hatching rate in artificial incubation. Eventually, higher percentage of fry survival could be obtained if there were increase in hatching rate. In this case, artificial incubators incapable to provide adequate space for egg incubation thus it reflected the main limiting factor that was responsible for the low hatching success. In conclusion, an increase in number of eggs in the artificial incubator would decrease in hatchability and result in lower percentage of fry production. Future research needs to develop an optimum artificial incubator in order to provide adequate space and aeration. Subsequently, better artificial incubator would increase the rate of hatching eggs and fry survival as well. Rana (1988) has already suggested that greater mechanical stress on the egg membrane may result in premature hatching. Therefore, it seems possible that the observed egg mortality was likely to have been associated with mechanical injuries making the eggs susceptible to bacterial or fungal infection (personal observations). Artificial incubation 91

92 systems for mouth-brooding tilapias should be designed to minimize mechanical stress to the eggs, which can be detrimental to their survival. One of the main advantages which artificial incubation offers is the possibility of reducing egg losses compared with maternal incubation (Yang and Kwak, 2004). However, in nature, oral incubation is a gentle and delicate process. Eventually, in this study, the eggs were kept in continuous circulation in artificial incubation. Although the incubators provide a somewhat smooth and gentle surface, this agitation could cause mechanical stress on the eggs which could result in physical damage to the eggs. 5.3 EFFECTS OF MATERNAL INCUBATION ON FRY SURVIVAL RATE WITH REFERENCE TO BODY SIZE (BODY WEIGHT AND BODY LENGTH) AND OVAPRIM INDUCTION (EXPERIMENT 3) Maternal incubation showed no significant differences in body weight and body length on fry survival rates. Ronald and Phelps (2005) found that the maternal incubation success rate was 55.0% and 91.3% for the Egypt and Ivory Coast strains. Successful maternal incubation also appears to be a learned trait where females in their first spawning season were less successful than those in their second season as evident by the Sagana and Lake Victoria strains that improved their incubation success by 81.2% and 112.2%, respectively. In the present study, short fish body length had the lowest fry survival rate compared with medium fish body length which exhibited the highest fry survival rate in maternal incubation. These differences may be due to fish age (as related to body size), history of previous spawning and with experience of incubating eggs which results in greatest incubation success. A major factor contributing to the difference in overall maternal spawning success may be the difference among strains and previous spawning history as related to picking back up eggs and successfully incubating them (Ronald and Phelps, 92

93 2005). The fry survival rate in maternal incubation in this study has been shown to be highly related to the percent hatched as well as the swim up of the fry. Reproductive parameters such as number of eggs, total weight of eggs, weight of one egg, diameter of eggs and hatching rate of eggs were not collected due to the physical limitation of the maternal incubation whereby the eggs were not disturbed to facilitate further studies (Experiment 3). In other words, the eggs incubated in its mouth were eventually hatched and the fry swam out naturally from the mouth. Furthermore, the brooding fish might be in a stressed condition and subsequently would end up with the egg bursting, cannibalism of eggs and even where the brooding fish would stop or not incubate all the eggs or none at all after sampling the eggs (Bolivar et al., 1993). Finally, this would produce a biased result in this experiment. Leornard (2001) also reported that eggs younger than 2 days post-spawning were soft and prone to rupturing. The material glair extruded with the eggs at the time of spawning might provide protection for the eggs in the first 2 days of incubation before they harden. 5.4 EFFECTS OF BODY SIZE (BODY WEIGHT AND BODY LENGTH) ON THE FRY SURVIVAL RATE FOR COMBINATION OF BOTH ARTIFICIAL AND MATERNAL INCUBATIONS (EXPERIMENTS 2 AND 3) The comparison of the effect between artificial and maternal incubations on the fry survival rates showed no apparent differences on weight of fish. However, body length was shown to significantly affect fry survival rates in both artificial incubation and maternal incubation. Some authors have considered the possibility that artificial incubation may equal or slightly improve the fry survival rate from maternal incubation under culture conditions (Evans et al., 1993). This is in agreement with the results of the present study. Shireman 93

94 (1991) reported that there were no significant effects of body size of tilapia fish on egg diameter, hatching rate and fry survival rate after using artificial incubation. In artificial and maternal incubation studies, Carral et al. (1988) obtained mean efficiency rates to juvenile stage 2 of 51.6% and 53.7% in signal crayfish eggs subjected to artificial and maternal incubation, respectively. With regard to this study, when maternal and artificial incubation were compared, the overall efficiency up to 2 weeks of fry survival from spawning were 94.89±0.69% and 94.95±1.39%, respectively. Therefore, the results of the current study confirmed that the fry survival rates were similar for both artificial and maternal incubations. In summary, our studies proved the efficacy of the tested system for the similarity between artificial and maternal incubations of tilapia fish (O.niloticus) on fry survival rates after 1.0 ml/kg ovaprim induction. In spite of numerous potential advantages of artificial incubation techniques, most researchers have affirmed the convenience of the eggs remaining brooded to the females throughout the embryonic development. However, artificial incubation has its merit for specific application as well as scientific studies of early embryonic development of tilapia fish. Therefore, detailed and refined experiments related to these aspects need to be conducted in the future. 94

95 Chapter GENERAL DISCUSSION Spawning induction by using commercially produced hormone such as ovaprim has been demonstrated to be effective in a variety of freshwater fishes (Zaini, 1994). In this study, the main objective was to evaluate the efficacy of ovaprim in red tilapia fish in an effort to improve reproductive characteristics such as number of eggs, total weight of eggs, weight of one egg, diameter of eggs, hatching rate and fry survival rate. It is well known that low fecundity and asynchronous spawning behaviour are the major constraints on tilapia seed production (Little and Hulata, 2000). Little application in spawning induction may be due to high cost of the ovaprim and hatchery operations, which prevent the implementation of such techniques by many fish farmers and to the relatively low importance of female-related infertility problems in red tilapia fish. However, in recent years, there has been an increase in the use of these techniques to evaluate egg quality, resulting in high correlations between several reproductive characteristics in artificial incubation systems of many fish species (Bhujel, 2000). These systems can provide accurate information on reproductive characteristics, although there is lack of uniformity among users. The present study on the red tilapia reproductive characteristics in relation to body size differences and artificial incubation system showed that large body size tilapia fish and medium body size tilapia fish produced higher number of eggs and total weight of eggs when compared to small body size tilapia fish (P<0.05). The reasons for body sizedependent variations in egg production and total weight of eggs might be a better positive response on ovaprim stimulations in medium and large body size tilapia fish. Higher production of E 2 in the blood was the positive stimulation prior to ovulation and spawning. 95

96 Hence, medium body size tilapia showed the highest E 2 levels followed by large and small body size tilapia. Correlations between ovaprim induction in plasma levels of gonadal steroids (E 2 ) and spawning were documented in a number of induced tilapia. During vitellogenesis, an increase in plasma E 2 levels, mainly 17β-oestradiol that correlates with the growth of vitellogenic oocytes has been observed in many species (Cornish, 1998). This has been proven when E 2 showed positive correlations between total weight of eggs and diameter of eggs, even though diameter of eggs has no significant effect on all body sizes of tilapia fish. The hatchability study in artificial incubation in the present study showed a negative correlation between numbers of eggs incubated. Higher number of eggs incubated in an artificial incubation would decrease in hatchability if compared with few numbers of eggs that were incubated in an artificial incubator. The physical characteristics of the eggs and the immobilization of the eggs within a limited space in an artificial incubator would be the limitation in hatching success. However, increase in hatchability would increase the fry survival rate (P<0.01). With reference to E 2 levels, it showed negative correlation with fry survival rate on the fish body weight but positive correlation on the fish body length. The reason for E 2 levels in fry survival is not known and never been reported. Therefore, further investigation is required in order to have better understanding of this parameter which may contribute towards determining the breeding efficacy of the red tilapia fish. In the current study, the main problems encountered in using ovaprim include the high investment costs and the difference in the standardization of dosage for spawning induction in red tilapia fish. This is because no available reports from previous authors regarding standard dosage of ovaprim stimulation for red tilapia fish was found. This was also explained by Rana (1988) about the flexibility and the absence of seasonal maturity and spawning for tilapia species. The broodstock selection in the present study showed 96

97 inaccuracy in confirming readiness of oocyte stage to undergo spawning induction. This might give negative results in certain ovaprim stimulations. The study about comparison between artificial and maternal incubation showed there were some limitations from the maternal incubation in evaluating number of eggs, total weight of eggs, weight of one egg, diameter of eggs and hatching rate. Some techniques must be designed to solve this problem by understanding the knowledge of brooding behaviour of this fish species in order to get appropriate data. By using only one type of artificial incubator, we were unable to compare different types of artificial incubator to elucidate the optimum conditions for the artificial incubation system. This suggestion is made due to the negative correlation between number of eggs and hatching rate obtained in this study. The results of the current study indicated that body weight had significant effect on egg production, but this evaluation was subjective due to highly coefficient correlation between body weights and body lengths. Reproductive characteristics might be influenced by numerous factors such as maturity levels, age, egg development stage, environmental conditions, spawning experience and the status of fish health. For E 2 analysis, high variation of E 2 even in the same group size after ovaprim stimulation was due to use of different fish at different times for serum collections. This was also probably due to the high flexibility of spawning behaviour in tilapia species (Rana and Macintosh, 1988). In order to improve E 2 evaluation in tilapia, new approaches should be applied, for example, collecting multiple serum at different time intervals from the same individual fish in the spawning induction programme. All the problems and limitations mentioned above could be minimized if the duration for this study was longer. However, this was not possible due to the designated duration for this study. Future studies may, therefore, allow more detailed investigation to 97

98 test the specific hormonal induction for tilapia species rather than ovaprim. Effect of fish age and stages of oocyte maturity could be the major factors for hormonal induction that could be prioritized in future studies. This will optimize egg production and characteristics, hatchability and fry survival in spawning induction. By studying spawning induction in red tilapia, it can consequently be a research model for other fish for spawning induction in artificial conditions. This study may serve as a catalyst for more comprehensive studies such as those involving molecular approaches for cloning and genetic manipulation. Applying egg characteristics, hatchability and fry survival in spawning induction may be important in the near future to provide cumulative improvement in the productivity of other fish species. In fish breeding programmes, accurate fertility evaluation and prediction are important in the selection of highly fertile fish at the lowest possible cost. This could help provide the human population with fish proteins, eggs or fry and other fishery products at reduced costs, and on a national scale, contribute to Malaysia s economy in becoming self-sufficient in food including fish and fish products. 98

99 Chapter CONCLUSIONS (a) A dose of 1.0 ml/kg body weight ovaprim exhibited a significantly higher response to spawning with reference to number of eggs per female (P<0.01) in medium and large body sizes compared to small body size in red tilapia fish. (b) Medium and large body weight broodstocks produced heavier eggs than small body weight broodstocks after 1.0 ml/kg ovaprim induction (P<0.01). However, weight of one egg produced was heavier in short body length broodstocks compared to long body length broodstocks. (c) Hatching rate was inversely related to the number of eggs per female incubated in an artificial incubator. This was due to space limitation which affected the egg incubation condition in the artificial incubator. (d) Increase in hatchability would increase the fry survival rate of red tilapia fish in artificial incubation (P<0.01). (e) No differences were found in fry survival rates between artificial incubation and maternal incubation. (f) Medium body size broodstocks produced the highest oestradiol (E 2 ) concentrations (P<0.05) and subsequently stimulating higher amount of eggs and total weight of eggs (P<0.01) in a single spawning. (g) Day 2 (spawning day) and during afternoon of days 1 to 3 exhibited the highest E 2 concentration for all sizes of broodstocks after 1.0 ml/kg ovaprim induction. 99

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108 APPENDICES APPENDIX 1: LIST OF MATERIALS Appendix Table 1.1: List of equipments, disposables and chemicals No. Item / Product Common name Commercial name Chemical name/ Content/Description Model/Catalogue Manufacture Local agent 1. Microscope Stereomicroscope Inverted light microscope 10x, 100x, 400x magnification (CK2) Olympus, USA (Borrowed equipment from ISB) 2. Weight balance - PB3002 Mettler-Toledo, Switzerland Chemopharm Sdn. Bhd. 3. Fibre tank 3 x 2 x 2 m³ - - Goodview Aquarium Sdn. Bhd. 4. Motor air pump Goodview Aquarium Sdn. Bhd. 5. Motor water pump Goodview Aquarium Sdn. Bhd. 6. Glass aquarium 2 x 1 x 1 m³ & 1 x ½ x ½ m³ - - Goodview Aquarium Sdn. Bhd. 7. Refrigerator - - Shel Lab Syarikat Bumi Sains 8. Artificial incubator Self made Fish holder Self made Fish net ½ x ½ cm² - - Goodview Aquarium Sdn. Bhd. 108

109 No. Item / Product Common name Commercial name Chemical name/ Content/Description Model/Catalogue Manufacture Local agent 11. Clamp cloth Goodmorning towel 1 dozen - - Goodview Aquarium Sdn. Bhd. 12. Rubber shoes 1 pair - - Power Hardware Sdn. Bhd. 13. Basin & pale 2 gallons - - Power Hardware Sdn. Bhd. 14. Water tube 1 diameter - - Goodview Aquarium Sdn. Bhd. 15. Air tube ¼ diameter - - Goodview Aquarium Sdn. Bhd. 16. Air stone 50 grams - - Goodview Aquarium Sdn. Bhd. 17. Spoon Power Hardware Sdn. Bhd. 18. Tally counter Interoffice Sdn. Bhd. 19. Stainless steel ruler 1 meter - - Interoffice Sdn. Bhd. 20. Terumo syringe & ½ needle 1cc/ml Tuberlin TERUMO Terumo, Philippine Intran marketing 21. Terumo syringe & 2 needle 5ml Tuberlin TERUMO Terumo, Philippine Intran marketing 22. Blood tube 6 ml vacutainer REF Becton Dickinson labware, USA Megalab Supplies Sdn. Bhd. 23. Plasma tip Disposable tips 1ml - Megalab Supplies Sdn. Bhd. 24. Plasma sucking tube Self made - - (Self-prepared) 25. Common salt 2 bags (50kg) - - Goodview Aquarium Sdn. Bhd. 109

110 No. Item / Product Common name Commercial name Chemical name/ Content/Description Model/Catalogue Manufacture Local agent 26. Antifungal Malachite green 240 ml - - Goodview Aquarium Sdn. Bhd. 27. Antichlorin 500g - - Goodview Aquarium Sdn. Bhd. 28. Fish pallet 20 kg Dindings Sdn. Bhd. Goodview Aquarium Sdn. Bhd. 29. GnRHa Ovaprim 10ml - Syndel International Inc., Canada 30. Micro-pipettor Eppendorf pipette 100 µl Eppendorf W. Germany 31. Water bath Memmert, Germany 32. Tips Disposable Syndel Asia Sdn. Bhd. Copens Scientific Malaysia Sdn. Bhd. Interscience Sdn. Bhd. Yellow - Megalab Supplies Sdn. Bhd. Megalab Supplies Sdn. Bhd. micropipette tips 33. Glove Disposable gloves Cross Protection Cross Protection (M) Sdn. Bhd. 110

111 Appendix Table 1.2: Standard calculation for ovaprim dosage preparation [Based on standard dosage of Ovaprim : 0.5 ml/kg] Example: Fish weight : 900 grams : 0.9 kg x 0.5 ml : 0.45 ml / 900g (given dosage) 1 st injection 0.45 ml x 20% 0.09 ml Cover for miss target 0.09 ml + 20% 0.09 ml ml ml 0.1 ml is the first dosage/injection. 2 nd injection 0.45 ml x 80% 0.36 ml Addition for miss target 0.36 ml x 20% 0.36 ml ml ml 0.4 ml is the second dosage/injection. 111

112 APPENDIX 2: LIST OF FIGURES FOR METHODOLOGY Appendix Figure 2.1: Ovaprim solution in a bottle with a syringe. Appendix Figure 2.2: 1 ml syringes for ovaprim injections. 112

113 Appendix Figure 2.3: Syringes loaded with ovaprim solution. Appendix Figure 2.4: 3 ml syringes for blood sampling. 113

114 Appendix Figure 2.5: Vacutainer tubes for blood centrifugation. Appendix Figure 2.6: 1 ml microcentrifuge serum tubes. 114

115 Appendix Figure 2.7: Storage of serum samples in a freezer. Appendix Figure 2.8: Polypropylene tubes (antibody-coated tubes) for E 2 count using the RIA technique. 115